Methods of producing promoter variants

ABSTRACT

A method of producing a protein of interest (POI) by culturing a recombinant eukaryotic cell line comprising an expression construct comprising a regulatable promoter and a nucleic acid molecule encoding a POI under the transcriptional control of said promoter, comprising the steps a) cultivating the cell line with a basal carbon source repressing the promoter, b) cultivating the cell line with a limited amount of a supplemental carbon source de-repressing the promoter to induce production of the POI at a transcription rate of at least 15% as compared to the native pGAP promoter, and c) producing and recovering the POI; and further an isolated regulatable promoter and a respective expression system.

The invention refers to regulatable promoters and a method of producinga protein of interest in a eukaryotic cell culture under the control ofa regulatable promoter.

BACKGROUND

Successful production of recombinant proteins has been accomplished witheukaryotic hosts. The most prominent examples are yeasts likeSaccharomyces cerevisiae, Pichia pastoris or Hansenula polymorpha,filamentous fungi like Aspergillus awamori or Trichoderma reesei, ormammalian cells like e.g. CHO cells. While the production of someproteins is readily achieved at high rates, many other proteins are onlyobtained at comparatively low levels.

The heterologous expression of a gene in a host organism usuallyrequires a vector allowing stable transformation of the host organism. Avector would provide the gene with a functional promoter adjacent to the5′ end of the coding sequence. The transcription is thereby regulatedand initiated by this promoter sequence. Most promoters used up to datehave been derived from genes that code for proteins that are usuallypresent at high concentrations in the cell.

EP0103409A2 discloses the use of yeast promoters associated withexpression of specific enzymes in the glycolytic pathway, i.e. promotersinvolved in expression of pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, phosphorglycerate mutase, hexokinase 1 and 2,glucokinase, phosphofructose kinase, aldolase and glycolytic regulationgene.

WO 97/44470 describes yeast promoters from Yarrowia lipolytica for thetranslation elongation factor 1 (TEF1) protein and for the ribosomalprotein S7 that are suitable for heterologous expression of proteins inyeast, and EP1951877A1 describes the use of the P. pastoris TEF1promoter for the production of heterologous proteins.

WO2005003310 provides methods for the expression of a coding sequence ofinterest in yeast using a promoter of the glyceraldehyde-3-phosphatedehydrogenase or phosphoglycerate mutase from oleaginous yeast Yarrowialipolytica.

Promoter sequences derived from genes involved in the methanol metabolicpathway of Pichia pastoris are disclosed in U.S. Pat. Nos. 4,808,537 and4,855,231 (alcohol oxidase AOX1, AOX2) and U.S. Pat. No. 6,730,499B1(formaldehyde dehydrogenase FLD1). US20080153126A1 includes mutantpromoter sequences based on the AOX1 promoter.

The AOX1 promoter is induced only in response to methanol and repressedby other carbon sources, such as glucose or ethanol. Methanol has thedisadvantage that it is unsuitable for use in the production of certainproducts, since it is potentially hazardous for its toxicity andflammability. Therefore, alternatives to the AOX1 promoter are sought.

US2008299616A1 introduces the regulatory sequences of the malatesynthase (MLS1) gene for heterologous gene expression in P. pastoris,which is repressed in media containing glucose and derepressed underglucose starvation conditions or when acetate is present. However, thissystem is not considered suitable for efficient production methods,since the MLS1 promoter is weak with low activity under de-repressedconditions.

Schöler and Schüller (Mol. Cell Biol. 1994 14(6):3613-22) describe thecontrol region of the isocitrate lyase gene ICL1, which is derepressedafter transfer of cells from fermentative to non-fermentative growthconditions.

WO2008063302A2 describes the use of novel inducible promoters derivedfrom ADH1 (alcohol dehydrogenase), ENO1 (enolase) and GUT1 genes of P.pastoris for the expression of heterologous proteins, CN1966688A the P.pastoris omega 3-fatty acid dehydrogenase promoter sequence, andWO002007117062A1 the P. pastoris derived auto-inducible NPS promoter,which is induced by phosphor limitation.

WO2008128701A2 describes the use of novel promoters, of which thepromoter derived from the TH13 (thiamine metabolism) gene of P. pastorisis repressed in medium containing thiamine, and derepressed uponthiamine depletion.

US2009325241 A1 describes a method of ethanol production in a yeast cellemploying a xylose-inducible promoter (FAS2 promoter).

It is desirable to provide improved recombinant eukaryotic cell lines toproduce fermentation products that can be isolated with high yields.Therefore, it is the object of the present invention to provide foralternative regulatory elements suitable for recombinant productionmethods, which are simple and efficient.

SUMMARY OF THE INVENTION

The object is solved by the subject matter as claimed.

According to the invention there is provided a method of producing aprotein of interest (POI) by culturing a recombinant eukaryotic cellline comprising an expression construct comprising a regulatablepromoter and a nucleic acid molecule encoding a POI under thetranscriptional control of said promoter, comprising the steps

a) cultivating the cell line with a basal carbon source repressing thepromoter,

b) cultivating the cell line with no or a limited amount of asupplemental carbon source de-repressing the promoter to induceproduction of the POI at a transcription rate of at least 15% ascompared to the native pGAP promoter of the cell, and

c) producing and recovering the POI.

Said cultivating steps specifically comprise cultivating the cell linein the presence of said carbon sources, thus, in a culture mediumcomprising said carbon sources, or in step b) also in the absence of asupplemental carbon source.

Said induction of POI production specifically refers to induction oftranscription, specifically including further translation and optionalexpression of said POI.

Said transcription rate specifically refers to the amount of transcriptsobtained upon fully inducing said promoter. Said promoter is consideredas de-repressed and fully induced, if the culture conditions provide forabout maximum induction, e.g. at glucose concentrations of less than 0.4g/L, preferably less than 0.04 g/L, specifically less than 0.02 g/L. Thefully induced promoter preferably shows a transcription rate of at least15%, preferably at least 20%, more preferred at least 30%, 40%, 50%,60%, 70%, 80%, 90% and at least 100% or even higher transcription rateof at least 150% or at least 200% as compared to the native pGAPpromoter. The transcription rate may, for example, be determined by theamount of transcripts of a reporter gene, such as eGFP, such asdescribed in the Example section below, which shows the relatively hightranscription rate of pG1 promoter of at least 50% as compared to thenative pGAP promoter, upon cultivating a clone in solution.Alternatively, the transcription rate may be determined by thetranscription strength on a microarray, where microarray data show thedifference of expression level between repressed and de-repressed stateand a high signal intensity in the fully induced state as compared tothe native pGAP promoter. Such microarray data specifically show atranscription rate of more than 200% for pG1, more than 30% for pG3 andpG4, more than 60% for pG6, more than 30% for pG7, more than 20% forpG8, each value as compared to the native pGAP. Prior art promoter MLS1or ICL1 were found to be too weak and thus not suitable for the purposeof the invention.

Said native pGAP promoter specifically is active in said recombinanteukaryotic cell in a similar way as in a native eukaryotic cell of thesame species or strain, including the unmodified (non-recombinant) orrecombinant eukaryotic cell. Such native pGAP promoter is commonlyunderstood to be an endogenous promoter, thus, homologous to theeukaryotic cell, and serves as a standard or reference promoter forcomparison purposes.

For example, a native pGAP promoter of P. pastoris is the unmodified,endogenous promoter sequence in P. pastoris, as used to control theexpression of GAPDH in P. pastoris, e.g. having the sequence shown inFIG. 13: native pGAP promoter sequence of P. pastoris (GS115) (SEQ ID13). If P. pastoris is used as a host for producing a POI according tothe invention, the transcription strength or rate of the promoteraccording to the invention is compared to such native pGAP promoter ofP. pastoris.

As another example, a native pGAP promoter of S. cerevisiae is theunmodified, endogenous promoter sequence in S. cerevisiae, as used tocontrol the expression of GAPDH in S. cerevisiae. If S. cerevisiae isused as a host for producing a POI according to the invention, thetranscription strength or rate of the promoter according to theinvention is compared to such native pGAP promoter of S. cerevisiae.

Therefore, the relative transcription strength or rate of a promoteraccording to the invention is usually compared to the native pGAPpromoter of a cell of the same species or strain that is used as a hostfor producing a POI.

According to a specific embodiment the basal carbon source is differentfrom the supplemental carbon source, e.g. quantitatively and/orqualitatively different. The quantitative difference may provide for thedifferent conditions to repress or de-repress the promoter activity.

According to a further specific embodiment the basal and thesupplemental carbon sources comprise the same type of molecules orcarbohydrates, preferably in different concentrations. According to afurther specific embodiment the carbon source is a mixture of two ormore different carbon sources.

Any type of organic carbon suitable used for eukaryotic cell culture maybe used. According to a specific embodiment the carbon source is ahexose such as glucose, fructose, galactose or mannose, a disaccharide,such as saccharose, an alcohol, such as glycerol or ethanol, or amixture thereof.

According to a specifically preferred embodiment, the basal carbonsource is selected from the group consisting of glucose, glycerol,ethanol, or mixtures thereof, and complex nutrient material. Accordingto a preferred embodiment, the basal carbon source is glycerol.

According to a further specific embodiment, the supplemental carbonsource is a hexose such as glucose, fructose, galactose and mannose, adisaccharide, such as saccharose, an alcohol, such as glycerol orethanol, or a mixture thereof. According to a preferred embodiment, thesupplemental carbon source is glucose.

Specifically, the method may employ glycerol as the basal carbon sourceand glucose as the supplemental carbon source.

The de-repressed conditions suitably may be achieved by specific means.Step b) optionally employs a feed medium that provides for no or thesupplemental carbon source in a limited amount.

Specifically, the feed medium is chemically defined and methanol-free.

The feed medium may be added to the culture medium in the liquid form orelse in an alternative form, such as a solid, e.g. as a tablet or othersustained release means, or a gas, e.g. carbon dioxide. Yet according toa preferred embodiment the limited amount of the supplemental carbonsource added to the cell culture medium, may even be zero. Preferably,the concentration of the supplemental carbon source in the culturemedium is 0-1 g/L, preferably less than 0.6 g/L, more preferred lessthan 0.3 g/L, more preferred less than 0.1 g/L, preferably 1-50 mg/L,more preferred 1-10 mg/L, specifically preferred 1 mg/L or even below,such as below the detection limit as measured with a suitable standardassay, e.g. determined as a residual concentration in the culture mediumupon consumption by the growing cell culture.

In a preferred method, the limited amount of the supplemental sourceprovides for a residual amount in the cell culture which is below thedetection limit as determined in the fermentation broth at the end of aproduction phase or in the output of a fermentation process, preferablyupon harvesting the fermentation product.

Preferably, the limited amount of the supplemental carbon source isgrowth limiting to keep the specific growth rate within the range of0.02 h⁻¹ to 0.2 h⁻¹, preferably 0.02 h⁻¹ to 0.15 h⁻¹.

According to a specific aspect of the invention, the promoter is aPichia pastoris promoter or a functionally active variant thereof.

Herein the promoter according to the invention shall always refer to thesequences described herein, and functionally active variants thereof. Asexplained in detail below, such variants include homologs and analogsderived from species other than Pichia pastoris.

The method according to the invention may employ a promoter which is awild-type promoter of P. pastoris or a functionally active variantthereof, e.g. capable of controlling the transcription of a specificgene in a wild-type or recombinant eukaryotic cell, e.g. a wild-typepromoter for selected genes, which gene is selected from the groupconsisting of G1 (SEQ ID 7), such as coding for a (high affinity)glucose transporter, G3 (SEQ ID 8), G4 (SEQ ID 9), such as coding for amitochondrial aldehyde dehydrogenase, G6 (SEQ ID 10), G7 (SEQ ID 11),such as coding for a member of the major facilitator sugar transporterfamily, or G8 (SEQ ID 12), such as coding for a member of the Gti1_Pac2superfamily, or a functionally active variant thereof.

According to the invention there is specifically provided a promoter ora functionally active variant thereof, which would be nativelyassociated with one of such genes in a wild-type yeast cell.

According to a specific embodiment, the cell line is selected from thegroup consisting of mammalian, insect, yeast, filamentous fungi andplant cell lines, preferably a yeast.

Specifically the yeast is selected from the group consisting of Pichia,Candida, Torulopsis, Arxula, Hensenula, Yarrowia, Kluyveromyces,Saccharomyces, Komagataella, preferably a methylotrophic yeast.

A specifically preferred yeast is Pichia pastoris, Komagataellapastoris, K. phaffii, or K. pseudopastoris.

According to a further specific embodiment, the promoter is not nativelyassociated with the nucleotide sequence encoding the POI.

Specifically, the POI is a eukaryotic protein, preferably a mammalianprotein.

A POI produced according to the invention may be a multimeric protein,preferably a dimer or tetramer.

According to one aspect of the invention, the POI is a recombinant orheterologous protein, preferably selected from therapeutic proteins,including antibodies or fragments thereof, enzymes and peptides, proteinantibiotics, toxin fusion proteins, carbohydrate—protein conjugates,structural proteins, regulatory proteins, vaccines and vaccine likeproteins or particles, process enzymes, growth factors, hormones andcytokines, or a metabolite of a POI.

A specific POI is an antigen binding molecule such as an antibody, or afragment thereof. Among specific POIs are antibodies such as monoclonalantibodies (mAbs), immunoglobulin (Ig) or immunoglobulin class G (IgG),heavy-chain antibodies (HcAb's), or fragments thereof such asfragment-antigen binding (Fab), Fd, single-chain variable fragment(scFv), or engineered variants thereof such as for example Fv dimers(diabodies), Fv trimers (triabodies), Fv tetramers, or minibodies andsingle-domain antibodies like VH or VHH or V-NAR.

According to a specific embodiment, a fermentation product ismanufactured using the POI, a metabolite or a derivative thereof.

According to another aspect of the invention, there is provided a methodfor controlling the expression of a POI in a recombinant eukaryotic cellunder the transcriptional control of a carbon source regulatablepromoter having a transcription strength of at least 15% as compared tothe native pGAP promoter of the cell, wherein the expression is inducedunder conditions limiting the carbon source. The carbon sourceregulatable promoter preferably has a transcription strength of at least20% as compared to the reference pGAP promoter, and specifically atranscription strength as described above with respect to thetranscription rate as compared to the native pGAP promoter. Therefore,the fully induced promoter preferably has a transcription strength of atleast 15%, preferably at least 20%, more preferred at least 30%, 40%,50%, 60%, 70%, 80%, 90% and at least 100% or an even highertranscription strength of at least 150% or at least 200% as compared tothe native pGAP promoter of the cell, as determined in the eukaryoticcell selected for producing the POI.

In a preferred embodiment such promoter is used that has atranscriptional activity or transcription strength in the de-repressedstate, which is at least 2 fold, more preferably at least 5 fold, evenmore preferred at least 10 fold, more preferred at least 20 fold, morepreferably at least 30, 40, 50, or 100 fold in the de-repressed statecompared to the repressed state.

According to another aspect of the invention, there is provided a methodof producing a POI in a recombinant eukaryotic cell under thetranscriptional control of a carbon source regulatable promoter, whereinsaid promoter has a transcription strength as described above, i.e. atleast 15% as compared to the native pGAP promoter of the cell. Thecarbon source regulatable promoter preferably has a transcriptionstrength of at least 20% as compared to the reference pGAP promoter,more preferred at least 30%, 40%, 50%, 60%, 70%, 80%, 90% and at least100% or an even higher transcription strength of at least 150% or atleast 200% as compared to the native pGAP promoter of the cell. In apreferred embodiment such promoter is used that has a transcriptionalactivity in the de-repressed state which is at least which is at least 2fold, more preferably at least 5 fold, even more preferred at least 10fold, more preferred at least 20 fold, more preferably at least 30, 40,50, or 100 fold in the de-repressed state compared to the repressedstate. Suitably a specific promoter according to the invention is usedin such a method.

In a specifically preferred method according to the invention, thepromoter is a the regulatable promoter comprising a nucleic acidsequence selected from the group consisting of

a) pG1 (SEQ ID 1), pG3 (SEQ ID 2), pG4 (SEQ ID 4), pG6 (SEQ ID 3), pG7(SEQ ID 5), or pG8 (SEQ ID 6);

b) a sequence having at least 60% homology to pG1 (SEQ ID 1), pG3 (SEQID 2), pG4 (SEQ ID 4), pG6 (SEQ ID 3), pG7 (SEQ ID 5), or pG8 (SEQ ID6);

c) a sequence which hybridizes under stringent conditions to pG1 (SEQ ID1), pG3 (SEQ ID 2), pG4 (SEQ ID 4), pG6 (SEQ ID 3), pG7 (SEQ ID 5), orpG8 (SEQ ID 6); and

d) a fragment or variant derived from a), b) or c),

wherein said promoter is a functionally active promoter, which is acarbon source regulatable promoter capable of expressing a POI in arecombinant eukaryotic cell at a transcription rate of at least 15% ascompared to the native pGAP promoter of the cell.

Specifically the variant of pG1 (SEQ ID 1), pG3 (SEQ ID 2), pG4 (SEQ ID4), pG6 (SEQ ID 3), pG7 (SEQ ID 5) or pG8 (SEQ ID 6) is a functionallyactive variant selected from the group consisting of homologs with atleast about 60% nucleotide sequence identity, homologs obtainable bymodifying the parent nucleotide sequence by insertion, deletion orsubstitution of one or more nucleotides within the sequence or at eitheror both of the distal ends of the sequence, preferably with a nucleotidesequence of at least 200 bp, preferably at least 250 bp, preferably atleast 300 bp, more preferred at least 400 bp, at least 500 bp, at least600 bp, at least 700 bp, at least 800 bp, at least 900 bp, or at least1000 bp, and analogs derived from species other than Pichia pastoris.

Some of the preferred functionally active variants of the promoteraccording to the invention are fragments of any of the pG1, pG3, pG4,pG6, pG7 or pG8 promoter nucleotide sequences, preferably fragmentsincluding the 3′ end of a promoter nucleotide sequence, e.g. anucleotide sequence derived from one of the promoter nucleotidesequences which has of a specific length and a deletion of the 5′terminal region, e.g. a cut-off of the nucleotide sequence at the 5′end, so to obtain a specific length with a range from the 3′ end to avarying 5′ end, such as with a length of the nucleotide sequence of atleast 200 bp, preferably at least 250 bp, preferably at least 300 bp,more preferred at least 400 bp, at least 500 bp, at least 600 bp, atleast 700 bp, at least 800 bp, at least 900 bp, or at least 1000 bp.

Exemplary variants have proven to be functionally active comprising orconsisting of such fragments, e.g. fragments with a specific lengthwithin the range of 200 to 1000 bp, preferably within the range of 250to 1000 bp, more preferably within the range of 300 to 1000 bp, e.g.including the 3′ terminal sequence. For example, a functionally activevariant of pG1 is selected from the group consisting of pG1a (SEQ ID41), pG1b (SEQ ID 42), pG1c (SEQ ID 43), pG1d (SEQ ID 44), pG1e (SEQ ID45) and pG1f (SEQ ID 46), thus, a nucleotide sequence within the rangeof 300-1000 bp, including the 3′ terminal sequence up to nucleotide1001.

According to another aspect of the invention, there is provided anisolated nucleic acid comprising a nucleic acid sequence selected fromthe group consisting of

a) pG1 (SEQ ID 1), pG3 (SEQ ID 2), pG6 (SEQ ID 3), pG7 (SEQ ID 5) or pG8(SEQ ID 6),

b) a sequence having at least 60% homology to pG1 (SEQ ID 1), pG3 (SEQID 2), pG6 (SEQ ID 3), pG7 (SEQ ID 5) or pG8 (SEQ ID 6),

c) a sequence which hybridizes under stringent conditions to pG1 (SEQ ID1), pG3 (SEQ ID 2), pG6 (SEQ ID 3), pG7 (SEQ ID 5) or pG8 (SEQ ID 6),and

d) a fragment or variant derived from a), b) or c),

wherein said nucleic acid comprises a functionally active promoter,which is a carbon source regulatable promoter capable of expressing aPOI in a recombinant eukaryotic cell at a transcription rate of at least15% as compared to the native pGAP promoter of the cell.

Specifically the variant of pG1 (SEQ ID 1), pG3 (SEQ ID 2), pG6 (SEQ ID3), pG7 (SEQ ID 5) or pG8 (SEQ ID 6) is a functionally active variantselected from the group consisting of homologs with at least about 60%nucleotide sequence identity, homologs obtainable by modifying theparent nucleotide sequence by insertion, deletion or substitution of oneor more nucleotides within the sequence or at either or both of thedistal ends of the sequence, preferably with a nucleotide sequence of atleast 200 bp, preferably at least 250 bp, preferably at least 300 bp,more preferred at least 400 bp, at least 500 bp, at least 600 bp, atleast 700 bp, at least 800 bp, at least 900 bp, or at least 1000 bp, andanalogs derived from species other than Pichia pastoris.

Some of the preferred functionally active variants of the promoteraccording to the invention are fragments of any of the pG1, pG3, pG6,pG7 or pG8 promoter nucleotide sequences, preferably fragments includingthe 3′ end of a promoter nucleotide sequence, e.g. a nucleotide sequencederived from one of the promoter nucleotide sequences which has of aspecific length and a deletion of the 5′ terminal region, e.g. a cut-offof the nucleotide sequence at the 5′ end, so to obtain a specific lengthwith a range from the 3′ end to a varying 5′ end, such as with a lengthof the nucleotide sequence of at least 200 bp, preferably at least 250bp, preferably at least 300 bp, more preferred at least 400 bp, at least500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900bp, or at least 1000 bp.

Exemplary variants have proven to be functionally active comprising orconsisting of such fragments, e.g. fragments with a specific lengthwithin the range of 200 to 1000 bp, preferably within the range of 250to 1000 bp, more preferably within the range of 300 to 1000 bp, e.g.including the 3′ terminal sequence. For example, a functionally activevariant of pG1 is selected from the group consisting of pG1a (SEQ ID41), pG1b (SEQ ID 42), pG1c (SEQ ID 43), pG1d (SEQ ID 44), pG1e (SEQ ID45) and pG1f (SEQ ID 46), thus, a nucleotide sequence within the rangeof 300-1000 bp, including the 3′ terminal sequence up to nucleotide1001.

The carbon source regulatable promoter preferably has a transcriptionstrength as described above, preferably at least 20% as compared to thereference pGAP promoter, more preferred at least 30%, 40%, 50%, 60%,70%, 80%, 90% and at least 100% or an even higher transcription strengthof at least 150% or at least 200% as compared to the native pGAPpromoter. In a preferred embodiment such promoter is used that has atranscriptional activity in the de-repressed state which is at leastwhich is at least 2 fold, more preferably at least 5 fold, even morepreferred at least 10 fold, more preferred at least 20 fold, morepreferably at least 30, 40, 50, or 100 fold in the de-repressed statecompared to the repressed state. Suitably a specific promoter accordingto the invention is used in such a method.

Yet, according to a further aspect of the invention, there is providedan expression construct comprising a promoter according to theinvention, operably linked to a nucleotide sequence encoding a POI underthe transcriptional control of said promoter, which promoter is notnatively associated with the coding sequence of the POI.

A further aspect of the invention refers to a vector comprising theconstruct according to the invention.

A further aspect of the invention refers to a recombinant eukaryoticcell comprising the construct or the vector according to the invention.

Specifically the cell is selected from the group consisting ofmammalian, insect, yeast, filamentous fungi and plant cell lines,preferably a yeast.

The yeast may suitably be selected from the group consisting of Pichia,Candida, Torulopsis, Arxula, Hensenula, Yarrowia, Kluyveromyces,Saccharomyces, Komagataella, preferably a methylotrophic yeast.

Preferably, the yeast is Pichia pastoris, Komagataella pastoris, K.phaffii, or K. pseudopastoris.

According to a specific embodiment a cell is employed, which has ahigher specific growth rate in the presence of a surplus of carbonsource relative to conditions of limited carbon source.

A further aspect of the invention refers to the use of the recombinanteukaryotic cell of the invention for the production of the POI.

According to a further aspect of the invention, there is provided amethod to screen or identify a carbon source regulatable promoter fromeukaryotic cells, comprising the steps of

a) cultivating eukaryotic cells in the presence of a carbon source in abatch culture under cell growing conditions,

b) further cultivating the cells in a fed batch culture in the presenceof a limited amount of a supplemental carbon source,

c) providing samples of the cell culture of step a) and b), and

d) performing transcription analysis in said samples to identify aregulatable promoter that shows a higher transcriptional strength incells of step b) than in cells of step a).

Said higher transcriptional strength may be determined by thetranscription strength in the fully induced state, which is e.g.obtained under conditions of glucose-limited chemostat cultivations,which is at least which is at least 2 fold, more preferably at least 5fold, even more preferred at least 10 fold, more preferred at least 20fold, more preferably at least 30, 40, 50, or 100 fold in thede-repressed state compared to the repressed state.

Preferably the transcription analysis is quantitative orsemi-quantitative, preferably employing DNA microarrays, RNA sequencingand transcriptome analysis.

FIGURES

FIG. 1: promoter sequence pG1 (SEQ ID 1) of P. pastoris.

FIG. 2: promoter sequence pG3 (SEQ ID 2) of P. pastoris.

FIG. 3: promoter sequence pG4 (SEQ ID 4) of P. pastoris.

FIG. 4: promoter sequence pG6 (SEQ ID 3) of P. pastoris.

FIG. 5: promoter sequence pG7 (SEQ ID 5) of P. pastoris.

FIG. 6: promoter sequence pG8 (SEQ ID 6) of P. pastoris.

FIG. 7: coding sequences of gene of GS115 genome 01 (SEQ ID 7) of P.pastoris.

FIG. 8: coding sequences of gene of GS115 genome G3 (SEQ ID 8) of P.pastoris.

FIG. 9: coding sequences of gene of GS115 genome G4 (SEQ ID 9) of P.pastoris.

FIG. 10: coding sequences of gene of GS115 genome G6 (SEQ ID 10) of P.pastoris.

FIG. 11: coding sequences of gene of GS115 genome G7 (SEQ ID 11) of P.pastoris.

FIG. 12: coding sequences of gene of GS115 genome G8 (SEQ ID 12) of P.pastoris.

FIG. 13: native pGAP promoter sequence of P. pastoris (GS115) (SEQ ID13)

GS115 # Name PAS* PIPA* description pGAP TDH3 PAS_chr2-1_0437 PIPA02510Glyceraldehyde- 3-phosphate dehydrogenase *PAS: ORF name in P. pastorisGS115; PIPA: ORF name in P. pastoris type strain DSMZ70382

FIG. 14: De-repression properties of the pG1 (circle), pG3 (triangle),pG4 (diamond) and pG6 (square) promoter: the maximum transcriptionactivity is reached for pG1 at around 0.04 g glucose/L or less, whereasall other pG promoters reach it already at around 4 g/L or less. Inorder to compare the relative induction behaviors of the differentpromoters, the data were normalized by dividing each value by the D20value of the respective promoter construct. Therefore the data arerelative fluorescence values, and the data points at D20 are 1.0.

FIG. 15: functionally active variants of the promoter sequence pG1;pG1a-f (SEQ ID 41-46) of P. pastoris.

DETAILED DESCRIPTION OF THE INVENTION

Specific terms as used throughout the specification have the followingmeaning.

The term “carbon source” as used herein shall mean a fermentable carbonsubstrate, typically a source carbohydrate, suitable as an energy sourcefor microorganisms, such as those capable of being metabolized by hostorganisms or production cell lines, in particular sources selected fromthe group consisting of monosaccharides, oligosaccharides,polysaccharides, alcohols including glycerol, in the purified form orprovided in raw materials, such as a complex nutrient material. Thecarbon source may be used according to the invention as a single carbonsource or as a mixture of different carbon sources.

A “basal carbon source” such as used according to the inventiontypically is a carbon source suitable for cell growth, such as anutrient for eukaryotic cells. The basal carbon source may be providedin a medium, such as a basal medium or complex medium, but also in achemically defined medium containing a purified carbon source. The basalcarbon source typically is provided in an amount to provide for cellgrowth, in particular during the growth phase in a cultivation process,for example to obtain cell densities of at least 5 g/L cell dry mass,preferably at least 10 g/L cell dry mass, or at least 15 g/L cell drymass, e.g. exhibiting viabilities of more than 90% during standardsub-culture steps, preferably more than 95%.

According to the invention the basal carbon source is typically used inan excess or surplus amount, which is understood as an excess providingenergy to increase the biomass, e.g. during the growth phase of a cellline in a fed-batch cultivation process. This surplus amount isparticularly in excess of the limited amount of a supplemental carbonsource to achieve a residual concentration in the fermentation broththat is measurable and typically at least 10 fold higher, preferably atleast 50 fold or at least 100 fold higher than during feeding thelimited amount of the supplemental carbon source.

The term “chemically defined” with respect to cell culture medium, suchas a feed medium in a fed-batch process, shall mean a growth mediumsuitable for the in vitro cell culture of a production cell line, inwhich all of the chemical components and peptides are known. Typically achemically defined medium is entirely free of animal-derived componentsand represents a pure and consistent cell culture environment.

A “supplemental carbon source” such as used according to the inventiontypically is a supplemental substrate facilitating the production offermentation products by production cell lines, in particular in theproduction phase of a cultivation process. The production phasespecifically follows a growth phase, e.g. in batch, fed-batch andcontinuous cultivation process. The supplemental carbon sourcespecifically may be contained in the feed of a fed-batch process.

A “limited amount” of a carbon source or a “limited carbon source” isherein understood as the amount of a carbon source necessary to keep aproduction cell line in a production phase or production mode. Such alimited amount may be employed in a fed-batch process, where the carbonsource is contained in a feed medium and supplied to the culture at lowfeed rates for sustained energy delivery to produce a POI, while keepingthe biomass at low growth rates. A feed medium is typically added to afermentation broth during the production phase of a cell culture.

The limited amount of the supplemental carbon source may, for example,be determined by the residual amount of the supplemental carbon sourcein the cell culture broth, which is below a predetermined threshold oreven below the detection limit as measured in a standard (carbohydrate)assay. The residual amount typically would be determined in thefermentation broth upon harvesting a fermentation product.

The limited amount of a supplemental carbon source may as well bedetermined by defining the average feed rate of the supplemental carbonsource to the fermenter, e.g. as determined by the amount added over thefull cultivation process, e.g. the fed batch phase, per cultivationtime, to determine a calculated average amount per time. This averagefeed rate is kept low to ensure complete usage of the supplementalcarbon source by the cell culture, e.g. between 0.6 g L⁻¹ h⁻¹ (g carbonsource per L initial fermentation volume and h time) and 25 g L⁻¹ h⁻¹,preferably between between 1.6 g L⁻¹ V and 20 g L⁻¹ V.

The limited amount of a supplemental carbon source may also bedetermined by measuring the specific growth rate before and during theproduction process, which specific growth rate is kept low during theproduction phase, e.g. within a predetermined range, such as in therange of 0.02 h⁻¹ to 0.20 h⁻¹, preferably between 0.02 h⁻¹ and 0.15 h⁻¹.

The term “cell line” as used herein refers to an established clone of aparticular cell type that has acquired the ability to proliferate over aprolonged period of time. The term “host cell line” refers to a cellline as used for expressing an endogenous or recombinant gene orproducts of a metabolic pathway to produce polypeptides or cellmetabolites mediated by such polypeptides. A “production host cell line”or “production cell line” is commonly understood to be a cell lineready-to-use for cultivation in a bioreactor to obtain the product of aproduction process, such as a POI. The term “eukaryotic host” or“eukaryotic cell line” shall mean any eukaryotic cell or organism, whichmay be cultivated to produce a POI or a host cell metabolite. It is wellunderstood that the term does not include human beings.

The term “expression” or “expression system” or “expression cassette”refers to nucleic acid molecules containing a desired coding sequenceand control sequences in operable linkage, so that hosts transformed ortransfected with these sequences are capable of producing the encodedproteins or host cell metabolites. In order to effect transformation,the expression system may be included in a vector; however, the relevantDNA may also be integrated into the host chromosome. Expression mayrefer to secreted or non-secreted expression products, includingpolypeptides or metabolites.

“Expression constructs” or “vectors” used herein are defined as DNAsequences that are required for the transcription of cloned recombinantnucleotide sequences, i.e. of recombinant genes and the translation oftheir mRNA in a suitable host organism. Expression vectors usuallycomprise an origin for autonomous replication in the host cells,selectable markers (e.g. an amino acid synthesis gene or a geneconferring resistance to antibiotics such as zeocin, kanamycin, G418 orhygromycin), a number of restriction enzyme cleavage sites, a suitablepromoter sequence and a transcription terminator, which components areoperably linked together. The terms “plasmid” and “vector” as usedherein include autonomously replicating nucleotide sequences as well asgenome integrating nucleotide sequences.

The term “variant” as used herein in the context of the presentinvention shall refer to any sequence with a specific homology oranalogy. The variant promoter may e.g. be derived from the promotersequence pG1 (SEQ ID 1), pG3 (SEQ ID 2), pG4 (SEQ ID 4), pG6 (SEQ ID 3),pG7 (SEQ ID 5) or pG8 (SEQ ID 6) by mutagenesis to produce sequencessuitable for use as a promoter in recombinant cell lines. Such variantpromoter may be obtained from a library of mutant sequences by selectingthose library members with predetermined properties. Variant promotersmay have the same or even improved properties, e.g. improved in inducingPOI production, with increased differential effect under repressing andde-repressing conditions. The variant promoter may also be derived fromanalogous sequences, e.g. from eukaryotic species other than Pichiapastoris or from a genus other than Pichia, such as from K. lactis, Z.rouxii, P. stipitis, H. polymorpha. Specifically, the analogous promotersequences natively associated with genes analogous to the correspondingP. pastoris genes may be used as such or as parent sequences to producefunctionally active variants thereof. Specifically,

-   -   a promoter analogous to pG1 is characterised that it is natively        associated with a gene analogous to G1 (high affinity glucose        transporter; P. pastoris GS115 description: Putative        transporter, member of the sugar porter family; coding sequence        SEQ ID 7);    -   a promoter analogous to pG3 is characterised that it is natively        associated with a gene analogous to G3 (coding sequence SEQ ID        8);    -   a promoter analogous to pG4 is characterised that it is natively        associated with a gene analogous to G4 (P. pastoris GS115:        predicted mitochondrial aldehyde dehydrogenase; coding sequence        SEQ ID 9);    -   a promoter analogous to pG6 is characterised that it is natively        associated with a gene analogous to G6 (coding sequence SEQ ID        10);    -   a promoter analogous to pG7 is characterised that it is natively        associated with a gene analogous to G7 (P. pastoris GS115:        member of the major facilitator sugar transporter family; coding        sequence SEQ ID 11);    -   a promoter analogous to pG8 is characterised that it is natively        associated with a gene analogous to G8 (P. pastoris GS115:        member of the Gti1_Pac2 superfamily; coding sequence SEQ ID 12).

The properties of such analogous promoter sequences or functionallyactive variants thereof may be determined using standard techniques.

The “functionally active” variant of a nucleotide or promoter sequenceas used herein means a sequence resulting from modification of a parentsequence by insertion, deletion or substitution of one or morenucleotides within the sequence or at either or both of the distal endsof the sequence, and which modification does not affect (in particularimpair) the activity of this sequence.

Specifically, the functionally active variant of the promoter sequenceaccording to the invention is selected from the group consisting of

-   -   homologs with at least about 60% nucleotide sequence identity,    -   homologs obtainable by modifying the parent nucleotide sequence        by insertion, deletion or substitution of one or more        nucleotides within the sequence or at either or both of the        distal ends of the sequence, preferably with (i.e. comprising or        consisting of) a nucleotide sequence of at least 200 bp,        preferably at least 300 bp, more preferred at least 400 bp, at        least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp,        at least 900 bp, or at least 1000 bp, and    -   analogs derived from species other than Pichia pastoris.

Specifically preferred functionally active variants are those derivedfrom a promoter according to the invention by modification and/orfragments of the promoter sequence, with (i.e. comprising or consistingof) a nucleotide sequence of at least 200 bp, preferably at least 250bp, preferably at least 300 bp, more preferred at least 400 bp, at least500 bp, at least 600 bp, at least 700 bp, at least 800 bp, at least 900bp, or at least 1000 bp.

Some of the preferred functionally active variants of the promoteraccording to the invention are fragments of any of the pG1, pG3, pG4,pG6, pG7 or pG8 promoter nucleotide sequences, preferably fragmentsincluding the 3′ end of a promoter nucleotide sequence, e.g. anucleotide sequence derived from one of the promoter nucleotidesequences which has of a specific length and a deletion of the 5′terminal region, e.g. a cut-off of the nucleotide sequence at the 5′end, so to obtain a specific length with a range from the 3′ end to avarying 5′ end, such as with a length of the nucleotide sequence of atleast 200 bp, preferably at least 250 bp, preferably at least 300 bp,more preferred at least 400 bp, at least 500 bp, at least 600 bp, atleast 700 bp, at least 800 bp, at least 900 bp, or at least 1000 bp.

Exemplary variants have proven to be functionally active comprising orconsisting of such fragments, e.g. fragments with a specific lengthwithin the range of 200 to 1000 bp, preferably within the range of 250to 1000 bp, more preferably within the range of 300 to 1000 bp, e.g.including the 3′ terminal sequence. For example, a functionally activevariant of pG1 is selected from the group consisting of pG1a (SEQ ID41), pG1b (SEQ ID 42), pG1c (SEQ ID 43), pG1d (SEQ ID 44), pG1e (SEQ ID45) and pG1f (SEQ ID 46), thus, a nucleotide sequence within the rangeof 300-1000 bp, including the 3′ terminal sequence up to nucleotide1001.

The term “regulatable” with respect to a promoter as used herein shallrefer to a promoter that is repressed in a eukaryotic cell in thepresence of an excess amount of a carbon source (nutrient substrate) inthe growth phase of a batch culture, and de-repressed to exert strongpromoter activity in the production phase of a production cell line,e.g. upon reduction of the amount of carbon, such as upon feeding of agrowth limiting carbon source (nutrient substrate) to a cultureaccording to the fed-batch strategy. In this regard, the term“regulatable” is understood as “carbon source-limit regulatable” or“glucose-limit regulatable”, referring to the de-repression of apromoter by carbon consumption, reduction, shortcoming or depletion, orby limited addition of the carbon source so that it is readily consumedby the cells.

The functionally active promoter according to the invention is arelatively strong regulatable promoter that is silenced or repressedunder cell growth conditions (growth phase), and activated orde-repressed under production condition (production phase), andtherefore suitable for inducing POI production in a production cell lineby limitating the carbon source. Therefore, the functionally activevariant of a promoter has at least such regulatable properties.

The strength of the regulatable promoter according to the inventionrefers to its transcription strength, represented by the efficiency ofinitiation of transcription occurring at that promoter with high or lowfrequency. The higher transcription strength the more frequentlytranscription will occur at that promoter. Promoter strength isimportant because it determines how often a given mRNA sequence istranscribed, effectively giving higher priority for transcription tosome genes over others, leading to a higher concentration of thetranscript. A gene that codes for a protein that is required in largequantities, for example, typically has a relatively strong promoter. TheRNA polymerase can only perform one transcription task at a time and somust prioritize its work to be efficient. Differences in promoterstrength are selected to allow for this prioritization. According to theinvention the regulatable promoter is relatively strong in the fullyinduced state, which is typically understood as the state of aboutmaximal activity. The relative strength is commonly determined withrespect to a standard promoter, such as the respective pGAP promoter ofthe cell as used as the host cell. The frequency of transcription iscommonly understood as the transcription rate, e.g. as determined by theamount of a transcript in a suitable assay, e.g. RT-PCR or Northernblotting. For example, the transcription strength of a promoteraccording to the invention is determined in the host cell which is P.pastoris and compared to the native pGAP promoter of P. pastoris.

The pGAP promoter initiates expression of the gap gene encodingglyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is aconstitutive promoter present in any microorganism capable of growing onglucose. GAPDH (EC 1\2\1\12), a key enzyme of glycolysis, plays acrucial role in catabolic and anabolic carbohydrate metabolism.

The regulatable promoter according to the invention exerts a relativelyhigh transcription strength, reflected by a transcription rate ortranscription strength of at least 15% as compared to the native pGAPpromoter in the host cell, sometimes called “homologous pGAP promoter”.Preferably the transcription rate or strength is at least 20%, inspecifically preferred cases at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, at least 90% and at least 100%or even higher, such as at least 150% or at least 200% as compared tothe native pGAP promoter, e.g. determined in the eukaryotic cellselected as host cell for producing the POI.

Specifically preferred is a regulatable promoter, which has in theinduced state at least a transcription strength of one of the pG1, pG3,PG4, pG6, pG7 or pG8 promoter. The comparative transcription strengthemploying the pGAP promoter as a reference may be determined by standardmeans, such as by measuring the quantity of transcripts, e.g. employinga microarray, or else in a cell culture, such as by measuring thequantity of respective gene expression products in recombinant cells. Anexemplary test is illustrated in the Examples section.

Specifically the promoter according to the invention is carbon sourceregulatable with a differential promoter strength as determined in atest comparing its strength in the presence of glucose and glucoselimitation, showing that it is still repressed at relatively highglucose concentrations, preferably at concentrations of at least 10 g/L,preferably at least 20 g/L. Specifically the promoter according to theinvention is fully induced at limited glucose concentrations and glucosethreshold concentrations fully inducing the promoter, which threshold isless than 20 g/L, preferably less than 10 g/L, less than 1 g/L, evenless than 0.1 g/L or less than 50 mg/L, preferably with a fulltranscription strength of e.g. at least 50% of the native, homologouspGAP promoter, at glucose concentrations of less than 40 mg/L.

Preferably the differential promoter strength is determined by theinitiation of POI production upon switching to inducing conditions belowa predetermined carbon source threshold, and compared to the strength inthe repressed state. The transcription strength commonly is understoodas the strength in the fully induced state, i.e. showing about maximumactivities under de-repressing conditions. The differential promoterstrength is, e.g. determined according to the efficiency or yield of POIproduction in a recombinant host cell line under de-repressingconditions as compared to repressing conditions, or else by the amountof a transcript. The regulatable promoter according to the invention hasa preferred differential promoter strength, which is at least 2 fold,more preferably at least 5 fold, even more preferred at least 10 fold,more preferred at least 20 fold, more preferably at least 30, 40, 50, or100 fold in the de-repressed state compared to the repressed state, alsounderstood as fold induction. Such differential promoter strength may bedetermined by a test as illustrated by the enclosed Examples.

Prior art promoter (MLS1 promoter or ICL1 promoter) turned out to have adifferential promoter strength of significantly less than the 2 foldinduction. Such prior art promoter was also not useful for industrialPOI production, with a promoter strength of around 5% as compared to thepGAP promoter standard. This has been proven in a direct comparison withthe promoter according to the invention.

The term “homology” indicates that two or more nucleotide sequences havethe same or conserved base pairs at a corresponding position, to acertain degree, up to a degree close to 100%. A homologous sequencetypically has at least about 50% nucleotide sequence identity,preferably at least about 60% identity, more preferably at least about70% identity, more preferably at least about 80% identity, morepreferably at least about 90% identity, more preferably at least about95% identity.

The homologous promoter sequence according to the invention preferablyhas a certain homology to any of the pG1, pG3, pG4, pG6, pG7 or pG8promoter nucleotide sequences of P. pastoris in at least specific partsof the nucleotide sequence, such as including the 3′ region of therespective promoter nucleotide sequence, preferably a part with aspecific length up to the 3′ end of the respective promoter nucleotidesequence, such as a part with a length of at least 200 bp, preferably atleast 250 bp, preferably at least 300 bp, more preferred at least 400bp, at least 500 bp, at least 600 bp, at least 700 bp, at least 800 bp,at least 900 bp, or at least 1000 bp, and analogs derived from speciesother than Pichia pastoris. Specifically at least those parts arepreferably homologous within the range of 300-1000 bp, including the 3′terminal sequence of the respective promoter nucleotide sequence.

Analogous sequences are typically derived from other species or strains.It is expressly understood that any of the analogous promoter sequencesof the present invention that are derived from species other than Pichiapastoris may comprise a homologous sequence, i.e. a sequence with acertain homology as described herein. Thus, the term “homologous” mayalso include analogous sequences. On the other hand, it is understoodthat the invention also refers to analogous sequences and homologsthereof that comprise a certain homology.

“Percent (%) identity” with respect to the nucleotide sequence of a geneis defined as the percentage of nucleotides in a candidate DNA sequencethat is identical with the nucleotides in the DNA sequence, afteraligning the sequence and introducing gaps, if necessary, to achieve themaximum percent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent nucleotide sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software. Those skilled in the art candetermine appropriate parameters for measuring alignment, including anyalgorithms needed to achieve maximal alignment over the full length ofthe sequences being compared.

The term “mutagenesis” as used in the context of the present inventionshall refer to a method of providing mutants of a nucleotide sequence,e.g. through insertion, deletion and/or substitution of one or morenucleotides, so to obtain variants thereof with at least one change inthe non-coding or coding region. Mutagenesis may be through random,semi-random or site directed mutation. Typically large randomized genelibraries are produced with a high gene diversity, which may be selectedaccording to a specifically desired genotype or phenotype.

The term “protein of interest (POI)” as used herein refers to apolypeptide or a protein that is produced by means of recombinanttechnology in a host cell. More specifically, the protein may either bea polypeptide not naturally occurring in the host cell, i.e. aheterologous protein, or else may be native to the host cell, i.e. ahomologous protein to the host cell, but is produced, for example, bytransformation with a self replicating vector containing the nucleicacid sequence encoding the POI, or upon integration by recombinanttechniques of one or more copies of the nucleic acid sequence encodingthe POI into the genome of the host cell, or by recombinant modificationof one or more regulatory sequences controlling the expression of thegene encoding the POI, e.g. of the promoter sequence. In some cases theterm POI as used herein also refers to any metabolite product by thehost cell as mediated by the recombinantly expressed protein.

The term “recombinant” as used herein shall mean “being prepared by orthe result of genetic engineering”. Thus, a “recombinant microorganism”comprises at least one “recombinant nucleic acid”. A recombinantmicroorganism specifically comprises an expression vector or cloningvector, or it has been genetically engineered to contain a recombinantnucleic acid sequence. A “recombinant protein” is produced by expressinga respective recombinant nucleic acid in a host. A “recombinantpromoter” is a genetically engineered non-coding nucleotide sequencesuitable for its use as a functionally active promoter as describedherein.

It surprisingly turned out that eukaryotic cells are capable of inducingthe production of a POI by limiting the availability of the carbonsource. Carbon starvation conditions were found to trigger induction ofstrong promoter activity, which was heretofore unknown in the art. TheMLS1 promoter of Pichia pastoris as described in US2008299616A1 to bederepressed under sugar limitations, was actually a comparably weakregulatable promoter for POI production. It was therefore surprisingthat such strong regulatable promoter of P. pastoris could be identifiedand could be used in eukaryotic production cell lines, in particular forrecombinant POI production.

Though the 9.43 Mbp genomic sequence of the GS115 strain of P. pastorishas been determined and disclosed in US20110021378A1, the properties ofindividual sequences, such as promoter sequences, have not beeninvestigated in detail. For instance, the pG4 sequence (SEQ ID 4) asdescribed herein was identified as a promoter sequence inUS20110021378A1, however, its regulatable properties or its use undercarbon starvation conditions were not known. It was even surprising thatsuch promoter could be effectively used in the method according to theinvention. Regulated promoters of the prior art such as used inindustrial scale POI production were mainly derived from the methanolmetabolic pathway and needed the addition of methanol to induce POIproduction, which is often not desired. The method according to theinvention has the advantage that it may provide for an increasedproduction by an enhanced expression, and has the reduced risk ofcontamination due to the specific promoter regulation, in particularwhen using a chemically defined medium, free of methanol.

It turned out that the regulatable promoter according to the inventionwould exert their regulatable activity only upon use of very specificculture media suitable for establishing promoter repressing andde-repressing conditions. As an example, P. pastoris could besuccessfully cultivated under conditions of an industrial productionprocess. First a batch culture on a basal carbon source, such asglycerol, was employed, followed by a fed batch with limited feed of asupplemental carbon source, such as glucose. Samples were taken close tothe end of the first batch phase, and in limited growth conditions, e.g.using a limited amount of supplemental carbon source. Transcriptomeanalysis with DNA micoarrays revealed specific genes that are stronglyactive on the supplemental carbon source and weak or inactive in thepresence of surplus carbon, i.e. the basal carbon source in excessamount. At least six promoter sequences were identified as regulatablepromoter according to the invention, i.e. pG1 (SEQ ID 1), pG3 (SEQ ID2), pG4 (SEQ ID 4), pG6 (SEQ ID 3), pG7 (SEQ ID 5) and pG8 (SEQ ID 6).The comparable MLS1 or ICL1 promoter of the prior art was only weak,with less than 1/10 of the strength of the pG1 promoter, and nodetectable regulation.

The features of repressing recombinant gene expression on the basalcarbon source, and strong expression on limited supplemental carbonsource, i.e. induction by substrate change could be verified infermentation processes.

The nucleotide sequences that could be used as regulatory sequencesaccording to the invention, which would provide for an improvedrecombinant protein production, can be obtained from a variety ofsources. The origin of the promoter according to the invention ispreferably from a yeast cell, most preferably from methylotrophic yeastsuch as from the Pichia genus or from the P. pastoris species, whichpromoter may then be used as a parent sequence to produce suitablevariants, e.g. mutants or analogs.

It is contemplated that a series of yeast cells, in particular of Pichiastrains, may be suitable to obtain respective promoter sequences thatare responsible for protein production under carbon starving conditions,or respective analogs in different species.

Variants of the identified P. pastoris promoter, including functionallyactive variants, such as homologs and analogs may be produced employingstandard techniques. The promoter may e.g. be modified to generatepromoter variants with altered expression levels and regulatoryproperties.

For instance, a promoter library may be prepared by mutagenesis of thepromoter sequences according to the invention, which may be used asparent molecules, e.g. to fine-tune the gene expression in eukaryoticcells by analysing variants for their expression under differentfermentation strategies and selecting suitable variants. A syntheticlibrary of variants may be used, e.g. to select a promoter matching therequirements for producing a selected POI. Such variants may haveincreased expression efficiency in eukaryotic host cells and highexpression upon depletion of a carbon source.

The differential fermentation strategies would distinguish between agrowth phase, such as step a) according to the present invention, and aproduction phase, such as step b).

Growth and/or production can suitably take place in batch mode,fed-batch mode or continuous mode. Any suitable bioreactor can be used,including batch, fed-batch, continuous, stirred tank reactor, or airliftreactor.

It is advantageous to provide for the fermentation process on a pilot orindustrial scale. The industrial process scale would preferably employvolumina of at least 10 L, specifically at least 50 L, preferably atleast 1 m³, preferably at least 10 m³, most preferably at least 100 m³.

Production conditions in industrial scale are preferred, which refer toe.g. fed batch cultivation in reactor volumes of 100 L to 10 m³ orlarger, employing typical process times of several days, or continuousprocesses in fermenter volumes of appr. 50-1000 L or larger, withdilution rates of approximately 0.02-0.15 h⁻¹.

The suitable cultivation techniques may encompass cultivation in abioreactor starting with a batch phase, followed by a short exponentialfed batch phase at high specific growth rate, further followed by a fedbatch phase at a low specific growth rate.

Another suitable cultivation technique may encompass a batch phasefollowed by a continuous cultivation phase at a low dilution rate.

A preferred embodiment of the invention includes a batch culture toprovide biomass followed by a fed-batch culture for high yields POIproduction.

For example, the cell line may be grown in step a) according to theinvention on glycerol or glucose to obtain biomass.

It is preferred to cultivate the host cell line according to theinvention in a bioreactor under growth conditions to obtain a celldensity of at least 1 g/L cell dry weight, more preferably at least 10g/L cell dry weight, preferably at least 20 g/L cell dry weight. It isadvantageous to provide for such yields of biomass production on a pilotor industrial scale.

A growth medium allowing the accumulation of biomass, specifically abasal growth medium, typically comprises a carbon source, a nitrogensource, a source for sulphur and a source for phosphate. Typically, sucha medium comprises furthermore trace elements and vitamins, and mayfurther comprise amino acids, peptone or yeast extract.

Preferred nitrogen sources include NH₄H₂PO₄, or NH₃ or (NH4)₂SO₄;

Preferred sulphur sources include MgSO₄, or (NH4)₂SO₄ or K₂SO₄;

Preferred phosphate sources include NH₄H₂PO₄, or H₃PO₄ or NaH₂PO₄,KH₂PO₄, Na₂HPO₄ or K₂HPO₄;

Further typical medium components include KCl, CaCl₂, and Trace elementssuch as: Fe, Co, Cu, Ni, Zn, Mo, Mn, I, B;

Preferably the medium is supplemented with vitamin B₇;

A typical growth medium for P. pastoris comprises glycerol or glucose,NH₄H₂PO₄, MgSO₄, KCl, CaCl₂, biotin, and trace elements.

In the production phase a production medium is specifically used withonly a limited amount of a supplemental carbon source.

Preferably the host cell line is cultivated in a mineral medium with asuitable carbon source, thereby further simplifying the isolationprocess significantly. An example of a preferred mineral medium is onecontaining an utilizable carbon source (e.g. glucose, glycerol ormethanol), salts containing the macro elements (potassium, magnesium,calcium, ammonium, chloride, sulphate, phosphate) and trace elements(copper, iodide, manganese, molybdate, cobalt, zinc, and iron salts, andboric acid), and optionally vitamins or amino acids, e.g. to complementauxotrophies.

The cells are cultivated under conditions suitable to effect expressionof the desired POI, which can be purified from the cells or culturemedium, depending on the nature of the expression system and theexpressed protein, e.g. whether the protein is fused to a signal peptideand whether the protein is soluble or membrane-bound. As will beunderstood by the skilled artisan, cultivation conditions will varyaccording to factors that include the type of host cell and particularexpression vector employed.

Induction of the POI production by the promoter according to theinvention is preferably controlled by cultivating the cells on asupplemental carbon source in a limited amount as the sole source ofcarbon and energy. The cells grow very slowly under carbon limitedconditions, but produce high yields of the POI under the control of theregulatable promoter.

The difference in the promoter activity specifically is at least 2 fold,preferably at least 5 fold, more preferred at least 10 fold, morepreferred at least 20 fold, more preferably at least 30, 40, 50, or 100fold in the de-repressed state compared to the repressed state.

By selecting the suitable promoter sequence according to the invention,optionally in combination with further preferred regulatory sequences,it is possible to provide for, under comparable conditions, at least thesame, or at least about a 1.5-fold, or at least about 2-fold, or atleast about a 5-fold, 10-fold, or at least up to about a 15-foldactivity as represented by the promoter activity or transcriptionstrength, or regulated by the promoter strength relative to a GAPpromoter that is homologous to the production cell, a native pGAP, orisolated from P. pastoris.

A typical production medium comprises the supplemental carbon source,and further NH₄H₂PO₄, MgSO₄, KCl, CaCl₂, biotin, and trace elements.

For example the feed of the supplemental carbon source added to thefermentation may comprise a carbon source with up to 50 wt % fermentablesugars. The low feed rate of the supplemental medium will limit theeffects of product inhibition on the cell growth, thus a high productyield based on substrate provision will be possible.

The fermentation preferably is carried out at a pH ranging from 3 to7.5.

Typical fermentation times are about 24 to 120 hours with temperaturesin the range of 20° C. to 35° C., preferably 22-30° C.

In general, the recombinant nucleic acids or organisms as referred toherein may be produced by recombination techniques well known to aperson skilled in the art. In accordance with the present inventionthere may be employed conventional molecular biology, microbiology, andrecombinant DNA techniques within the skill of the art. Such techniquesare explained fully in the literature. See, e.g., Maniatis, Fritsch &Sambrook, “Molecular Cloning: A Laboratory Manual (1982).

According to a preferred embodiment of the present invention, arecombinant construct is obtained by ligating the promoter and relevantgenes into a vector. These genes can be stably integrated into the hostcell genome by transforming the host cell using such vectors.

Expression vectors may include but are not limited to cloning vectors,modified cloning vectors and specifically designed plasmids. Thepreferred expression vector as used in the invention may be anyexpression vector suitable for expression of a recombinant gene in ahost cell and is selected depending on the host organism. Therecombinant expression vector may be any vector which is capable ofreplicating in or integrating into the genome of the host organisms,also called host vector.

In the present invention, it is preferred to use plasmids derived frompPUZZLE as the vector.

Appropriate expression vectors typically comprise further regulatorysequences suitable for expressing DNA encoding a POI in a eukaryotichost cell. Examples of regulatory sequences include operators,enhancers, ribosomal binding sites, and sequences that controltranscription and translation initiation and termination. The regulatorysequences may be operably linked to the DNA sequence to be expressed.

To allow expression of a recombinant nucleotide sequence in a host cell,the expression vector may provide the promoter according to theinvention adjacent to the 5′ end of the coding sequence, e.g. upstreamfrom a signal peptide gene. The transcription is thereby regulated andinitiated by this promoter sequence.

A signal peptide may be a heterologous signal peptide or a hybrid of anative and a heterologous signal peptide, and may specifically beheterologous or homologous to the host organism producing the protein.The function of the signal peptide is to allow the POI to be secreted toenter the endoplasmatic reticulum. It is usually a short (3-60 aminoacids long) peptide chain that directs the transport of a proteinoutside the plasma membrane, thereby making it easy to separate andpurify the heterologous protein. Some signal peptides are cleaved fromthe protein by signal peptidase after the proteins are transported.

Exemplary signal peptides are signal sequences from S. cerevisiaealpha-mating factor prepro peptide and the signal peptide from the P.pastoris acid phosphatase gene (PHO1).

A promoter sequence is understood to be operably linked to a codingsequence, if the promotor controls the transcription of the codingsequence. If a promoter sequence is not natively associated with thecoding sequence, its transcription is either not controlled by thepromoter in native (wild-type) cells or the sequences are recombinedwith different contiguous sequences.

To prove the function of the relevant sequences, expression vectorscomprising one or more of the regulatory elements may be constructed todrive expression of a POI, and the expressed yield is compared toconstructs with conventional regulatory elements. A detailed descriptionof the experimental procedure can be found in the examples below. Theidentified genes may be amplified by PCR from P. pastoris using specificnucleotide primers, cloned into an expression vector and transformedinto a eukaryotic cell line, e.g. using a yeast vector and a strain ofP. pastoris, for high level production of various different POI. Toestimate the effect of the promoter according to the invention on theamount of recombinant POI so produced, the eukaryotic cell line may becultured in shake flask experiments and fedbatch or chemostatfermentations in comparison with strains comprising a conventional, noncarbon source regulatable promoter, such as for example the standardpGAP promoter in the respective cell. In particular, the choice of thepromoter has a great impact on the recombinant protein production.

Preferred methods of transformation for the uptake of the recombinantDNA fragment by the microorganism include chemical transformation,electroporation or transformation by protoplastation. Transformantsaccording to the present invention can be obtained by introducing such avector DNA, e.g. plasmid DNA, into a host and selecting transformantswhich express the relevant protein or host cell metabolite with highyields.

The POI can be produced using the recombinant host cell line byculturing a transformant, thus obtained in an appropriate medium,isolating the expressed product or metabolite from the culture, andoptionally purifying it by a suitable method.

Transformants according to the present invention can be obtained byintroducing such a vector DNA, e.g. plasmid DNA, into a host andselecting transformants which express the POI or the host cellmetabolite with high yields. Host cells are treated to enable them toincorporate foreign DNA by methods conventionally used fortransformation of eukaryotic cells, such as the electric pulse method,the protoplast method, the lithium acetate method, and modified methodsthereof. P. pastoris is preferably transformed by electroporation.

The preferred host cell line according to the invention maintains thegenetic properties employed according to the invention, and theproduction level remains high, e.g. at least at a μg level, even afterabout 20 generations of cultivation, preferably at least 30 generations,more preferably at least 40 generations, most preferred of at least 50generations. The stable recombinant host cell is considered a greatadvantage when used for industrial scale production.

Several different approaches for the production of the POI according tothe method of the invention are preferred. Substances may be expressed,processed and optionally secreted by transforming a eukaryotic host cellwith an expression vector harbouring recombinant DNA encoding a relevantprotein and at least one of the regulatory elements as described above,preparing a culture of the transformed cell, growing the culture,inducing transcription and POI production, and recovering the product ofthe fermentation process.

The POI is preferably expressed employing conditions to produce yieldsof at least 1 mg/L, preferably at least 10 mg/L, preferably at least 100mg/L, most preferred at least 1 g/L.

The host cell according to the invention is preferably tested for itsexpression capacity or yield by the following test: ELISA, activityassay, H PLC, or other suitable tests.

It is understood that the methods disclosed herein may further includecultivating said recombinant host cells under conditions permitting theexpression of the POI, preferably in the secreted form or else asintracellular product. A recombinantly produced POI or a host cellmetabolite can then be isolated from the cell culture medium and furtherpurified by techniques well known to a person skilled in the art.

The POI produced according to the invention typically can be isolatedand purified using state of the art techniques, including the increaseof the concentration of the desired POI and/or the decrease of theconcentration of at least one impurity.

If the POI is secreted from the cells, it can be isolated and purifiedfrom the culture medium using state of the art techniques. Secretion ofthe recombinant expression products from the host cells is generallyadvantageous for reasons that include facilitating the purificationprocess, since the products are recovered from the culture supernatantrather than from the complex mixture of proteins that results when yeastcells are disrupted to release intracellular proteins.

The cultured transformant cells may also be ruptured sonically ormechanically, enzymatically or chemically to obtain a cell extractcontaining the desired POI, from which the POI is isolated and purified.

As isolation and purification methods for obtaining a recombinantpolypeptide or protein product, methods, such as methods utilizingdifference in solubility, such as salting out and solvent precipitation,methods utilizing difference in molecular weight, such asultrafiltration and gel electrophoresis, methods utilizing difference inelectric charge, such as ion-exchange chromatography, methods utilizingspecific affinity, such as affinity chromatography, methods utilizingdifference in hydrophobicity, such as reverse phase high performanceliquid chromatography, and methods utilizing difference in isoelectricpoint, such as isoelectric focusing may be used.

The highly purified product is essentially free from contaminatingproteins, and preferably has a purity of at least 90%, more preferred atleast 95%, or even at least 98%, up to 100%. The purified products maybe obtained by purification of the cell culture supernatant or else fromcellular debris.

As isolation and purification methods the following standard methods arepreferred: Cell disruption (if the POI is obtained intracellularly),cell (debris) separation and wash by Microfiltration or Tangential FlowFilter (TFF) or centrifugation, POI purification by precipitation orheat treatment, POI activation by enzymatic digest, POI purification bychromatography, such as ion exchange (IEX), hydrophobic ointeractionchromatography (HIC), Affinity chromatography, size exclusion (SEC) orHPLC Chromatography, POI precipitation of concentration and washing byultrafiltration steps.

The isolated and purified POI can be identified by conventional methodssuch as Western blot, HPLC, activity assay, or ELISA.

The POI can be any eukaryotic, prokaryotic or synthetic polypeptide. Itcan be a secreted protein or an intracellular protein. The presentinvention also provides for the recombinant production of functionalhomologs, functional equivalent variants, derivatives and biologicallyactive fragments of naturally occurring proteins. Functional homologsare preferably identical with or correspond to and have the functionalcharacteristics of a sequence.

A POI referred to herein may be a product homologous to the eukaryotichost cell or heterologous, preferably for therapeutic, prophylactic,diagnostic, analytic or industrial use.

The POI is preferably a heterologous recombinant polypeptide or protein,produced in a eukaryotic cell, preferably a yeast cell, preferably assecreted proteins. Examples of preferably produced proteins areimmunoglobulins, immunoglobulin fragments, aprotinin, tissue factorpathway inhibitor or other protease inhibitors, and insulin or insulinprecursors, insulin analogues, growth hormones, interleukins, tissueplasminogen activator, transforming growth factor a or b, glucagon,glucagon-like peptide 1 (GLP-1), glucagon-like peptide 2 (GLP-2), GRPP,Factor VII, Factor VIII, Factor XIII, platelet-derived growth factor1,serum albumin, enzymes, such as lipases or proteases, or a functionalhomolog, functional equivalent variant, derivative and biologicallyactive fragment with a similar function as the native protein. The POImay be structurally similar to the native protein and may be derivedfrom the native protein by addition of one or more amino acids to eitheror both the C- and N-terminal end or the side-chain of the nativeprotein, substitution of one or more amino acids at one or a number ofdifferent sites in the native amino acid sequence, deletion of one ormore amino acids at either or both ends of the native protein or at oneor several sites in the amino acid sequence, or insertion of one or moreamino acids at one or more sites in the native amino acid sequence. Suchmodifications are well known for several of the proteins mentionedabove.

A POI can also be selected from substrates, enzymes, inhibitors orcofactors that provide for biochemical reactions in the host cell, withthe aim to obtain the product of said biochemical reaction or a cascadeof several reactions, e.g. to obtain a metabolite of the host cell.Exemplary products can be vitamins, such as riboflavin, organic acids,and alcohols, which can be obtained with increased yields following theexpression of a recombinant protein or a POI according to the invention.

In general, the host cell, which expresses a recombinant product, can beany eukaryotic cell suitable for recombinant expression of a POI.

Examples of preferred mammalian cells are BHK, CHO (CHO-DG44,CHO-DUXB11, CHO-DUKX, CHO-K1, CHOK1 SV, CHO-S), HeLa, HEK293, MDCK,NIH3T3, NS0, PER.C6, SP2/0 and VERO cells.

Examples of preferred yeast cells used as host cells according to theinvention include but are not limited to the Saccharomyces genus (e.g.Saccharomyces cerevisiae), the Pichia genus (e.g. P. pastoris, or P.methanolica), the Komagataella genus (K. pastoris, K. pseudopastoris orK. phaffii), Hansenula polymorpha or Kluyveromyces lactis.

Newer literature divides and renames Pichia pastoris into Komagataellapastoris, Komagataella phaffii and Komagataella pseudopastoris. HereinPichia pastoris is used synonymously for all, Komagataella pastoris,Komagataella phaffii and Komagataella pseudopastoris.

The preferred yeast host cells are derived from methylotrophic yeast,such as from Pichia or Komagataella, e.g. Pichia pastoris, orKomagataella pastoris, or K. phaffii, or K. pseudopastoris. Examples ofthe host include yeasts such as P. pastoris. Examples of P. pastorisstrains include CBS 704 (=NRRL Y-1603=DSMZ 70382), CBS 2612 (=NRRLY-7556), CBS 7435 (=NRRL Y-11430), CBS 9173-9189 (CBS strains: CBS-KNAWFungal Biodiversity Centre, Centraalbureau voor Schimmelcultures,Utrecht, The Netherlands), and DSMZ 70877 (German Collection ofMicroorganisms and Cell Cultures), but also strains from Invitrogen,such as X-33, GS115, KM71 and SMD1168. Examples of S. cerevisiae strainsinclude W303, CEN.PK and the BY-series (EUROSCARF collection). All ofthe strains described above have been successfully used to producetransformants and express heterologous genes.

A preferred yeast host cell according to the invention, such as a P.pastoris or S. cerevisiae host cell, contains a heterologous orrecombinant promoter sequences, which may be derived from a P. pastorisor S. cerevisiae strain, different from the production host. In anotherspecific embodiment the host cell according to the invention comprises arecombinant expression construct according to the invention comprisingthe promoter originating from the same genus, species or strain as thehost cell.

The promoter may be a promoter according to the invention or any otherDNA sequence which shows transcriptional activity in the host cell andmay be derived from genes encoding proteins either homologous orheterologous to the host. The promoter is preferably derived from a geneencoding a protein homologous to the host cell.

For example, a promoter according to the invention may be derived fromyeast, such as a S. cerevisiae strain, and be used to express a POI in ayeast. A specifically preferred embodiment relates to a promoteraccording to the invention originating from P. pastoris for use in amethod to produce a recombinant POI in a P. pastoris producer host cellline. The homologous origin of the nucleotide sequence facilitates itsincorporation into the host cell of the same genus or species, thusenabling stable production of a POI, possibly with increased yields inindustrial manufacturing processes. Also, functionally active variantsof the promoter from other suitable yeasts or other fungi or from otherorganisms such as vertebrates or plants can be used.

If the POI is a protein homologous to the host cell, i.e. a proteinwhich is naturally occurring in the host cell, the expression of the POIin the host cell may be modulated by the exchange of its native promotersequence with a promoter sequence according to the invention.

This purpose may be achieved e.g. by transformation of a host cell witha recombinant DNA molecule comprising homologous sequences of the targetgene to allow site specific recombination, the promoter sequence and aselective marker suitable for the host cell. The site specificrecombination shall take place in order to operably link the promotersequence with the nucleotide sequence encoding the POI. This results inthe expression of the POI from the promoter sequence according to theinvention instead of from the native promoter sequence.

In a specifically preferred embodiment of the invention the promotersequence has an increased promoter activity relative to the nativepromoter sequence of the POI.

According to the invention it is preferred to provide a P. pastoris hostcell line comprising a promoter sequence according to the inventionoperably linked to the nucleotide sequence coding for the POI.

According to the invention it is also possible to provide a wildcardvector or host cell according to the invention, which comprises apromoter according to the invention, and which is ready to incorporate agene of interest encoding a POI. The wildcard cell line is, thus, apreformed host cell line, which is characterized for its expressioncapacity. This follows an innovative “wildcard” platform strategy forthe generation of producer cell lines, for the POI production, e.g.using site-specific recombinase-mediated cassette exchange. Such a newhost cell facilitates the cloning of a gene of interest (GOI), e.g. intopredetermined genomic expression hot spots within days in order to getreproducible, highly efficient production cell lines.

According to a preferred embodiment the method according to theinvention employs a recombinant nucleotide sequence encoding the POI,which is provided on a plasmid suitable for integration into the genomeof the host cell, in a single copy or in multiple copies per cell. Therecombinant nucleotide sequence encoding the POI may also be provided onan autonomously replicating plasmid in a single copy or in multiplecopies per cell.

The preferred method according to the invention employs a plasmid, whichis a eukaryotic expression vector, preferably a yeast expression vector.Expression vectors may include but are not limited to cloning vectors,modified cloning vectors and specifically designed plasmids. Thepreferred expression vector as used in the invention may be anyexpression vector suitable for expression of a recombinant gene in ahost cell and is selected depending on the host organism. Therecombinant expression vector may be any vector which is capable ofreplicating in or integrating into the genome of the host organisms,also called host vector, such as a yeast vector, which carries a DNAconstruct according to the invention. A preferred yeast expressionvector is for expression in yeast selected from the group consisting ofmethylotrophic yeasts represented by the genera Hansenula, Pichia,Candida and Torulopsis.

In the present invention, it is preferred to use plasmids derived frompPICZ, pGAPZ, pPIC9, pPICZalfa, pGAPZalfa, pPIC9K, pGAPHis or pPUZZLE asthe vector.

According to a preferred embodiment of the present invention, arecombinant construct is obtained by ligating the relevant genes into avector. These genes can be stably integrated into the host cell genomeby transforming the host cell using such vectors. The polypeptidesencoded by the genes can be produced using the recombinant host cellline by culturing a transformant, thus obtained in an appropriatemedium, isolating the expressed POI from the culture, and purifying itby a method appropriate for the expressed product, in particular toseparate the POI from contaminating proteins.

Expression vectors may comprise one or more phenotypic selectablemarkers, e.g. a gene encoding a protein that confers antibioticresistance or that supplies an autotrophic requirement. Yeast vectorscommonly contain an origin of replication from a yeast plasmid, anautonomously replicating sequence (ARS), or alternatively, a sequenceused for integration into the host genome, a promoter region, sequencesfor polyadenylation, sequences for transcription termination, and aselectable marker.

The procedures used to ligate the DNA sequences, e.g. coding for theprecursing sequence and/or the POI, the promoter and the terminator,respectively, and to insert them into suitable vectors containing theinformation necessary for integration or host replication, are wellknown to persons skilled in the art, e.g. described by J. Sambrook etal., “Molecular Cloning 2nd ed.”, Cold Spring Harbor Laboratory Press(1989).

It will be understood that the vector, which uses the regulatoryelements according to the invention and/or the POI as an integrationtarget, may be constructed either by first preparing a DNA constructcontaining the entire DNA sequence coding for the regulatory elementsand/or the POI and subsequently inserting this fragment into a suitableexpression vector, or by sequentially inserting DNA fragments containinggenetic information for the individual elements, such as the signal,leader or heterologous protein, followed by ligation.

Also multicloning vectors, which are vectors having a multicloning site,can be used according to the invention, wherein a desired heterologousgene can be incorporated at a multicloning site to provide an expressionvector. In expression vectors, the promoter is placed upstream of thegene of the POI and regulates the expression of the gene. In the case ofmulticloning vectors, because the gene of the POI is introduced at themulticloning site, the promoter is placed upstream of the multicloningsite.

The DNA construct as provided to obtain a recombinant host cellaccording to the invention may be prepared synthetically by establishedstandard methods, e.g. the phosphoramidite method. The DNA construct mayalso be of genomic or cDNA origin, for instance obtained by preparing agenomic or cDNA library and screening for DNA sequences coding for allor part of the polypeptide of the invention by hybridization usingsynthetic oligonucleotide probes in accordance with standard techniques(Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor, 1989). Finally, the DNA construct may be of mixed synthetic andgenomic, mixed synthetic and cDNA or mixed genomic and cDNA originprepared by annealing fragments of synthetic, genomic or cDNA origin, asappropriate, the fragments corresponding to various parts of the entireDNA construct, in accordance with standard techniques.

In another preferred embodiment, the yeast expression vector is able tostably integrate in the yeast genome, e. g. by homologous recombination.

A transformant host cell according to the invention obtained bytransforming the cell with the regulatory elements according to theinvention and/or the POI genes may preferably first be cultivated atconditions to grow efficiently to a large cell number without the burdenof expressing a heterologous protein. When the cell line is prepared forthe POI expression, cultivation techniques are chosen to produce theexpression product.

The subject matter of the following definitions is consideredembodiments of the present invention:

1. A method of producing a protein of interest (POI) by culturing arecombinant eukaryotic cell line comprising an expression constructcomprising a regulatable promoter and a nucleic acid molecule encoding aPOI under the transcriptional control of said promoter, comprising thesteps

a) cultivating the cell line with a basal carbon source repressing thepromoter,

b) cultivating the cell line with no or a limited amount of asupplemental carbon source de-repressing the promoter to induceproduction of the POI at a transcription rate of at least 15% ascompared to the native pGAP promoter of the cell, and

c) producing and recovering the POI.

2. Method according to definition 1, wherein the basal carbon source isselected from the group consisting of glucose, glycerol, ethanol andcomplex nutrient material.

3. Method according to definition 1 or 2, wherein the supplementalcarbon source is a hexose such as glucose, fructose, galactose ormannose, a disaccharide, such as saccharose, an alcohol, such asglycerol or ethanol, or a mixture thereof.

4. Method according to any of definitions 1 to 3, wherein the basalcarbon source is glycerol, and the supplemental carbon source isglucose.

5. Method according to any of definitions 1 to 4, wherein step b)employs a feed medium that provides for no or the supplemental carbonsource in a limited amount, preferably 0-1 g/L in the culture medium.

6. Method according to definition 5, wherein the feed medium ischemically defined and methanol-free.

7. Method according to any of definitions 1 to 6, wherein the limitedamount of the supplemental carbon source is growth limiting to keep thespecific growth rate within the range of 0.02 h⁻¹ to 0.2 h⁻¹, preferably0.02 h⁻¹ to 0.15 h⁻¹.

8. Method according to definition 7, wherein the limited amount of thesupplemental source provides for a residual amount in the cell culturewhich is below the detection limit.

9. Method according to any of definitions 1 to 8, wherein the promoteris capable of controlling the transcription of a gene in a wild-typeeukaryotic cell, which gene is selected from the group consisting of G1(SEQ ID 7), G3 (SEQ ID 8), G4 (SEQ ID 9), G6 (SEQ ID 10), G7 (SEQ ID 11)or G8 (SEQ ID 12), or a functionally active variant thereof.

10. Method according to definition 9, wherein said functionally activevariants are selected from the group consisting of homologs with atleast about 60% nucleotide sequence identity, homologs obtainable bymodifying the parent nucleotide sequence by insertion, deletion orsubstitution of one or more nucleotides within the sequence or at eitheror both of the distal ends of the sequence, preferably comprising orconsisting of a nucleotide sequence of at least 200 bp, and analogsderived from species other than Pichia pastoris.

11. Method according to definition 9 or 10, wherein the functionallyactive variant of pG1 is selected from the group consisting of pG1a (SEQID 41), pG1b (SEQ ID 42), pG1c (SEQ ID 43), pG1d (SEQ ID 44), pG1e (SEQID 45) and pG1f (SEQ ID 46).

12. Method according to any of definitions 1 to 11, wherein the promoteris a Pichia pastoris promoter or a functionally active variant thereof.

13. Method according to any of definitions 1 to 12, wherein the cellline is selected from the group consisting of mammalian, insect, yeast,filamentous fungi and plant cell lines, preferably a yeast.

14. Method according to definition 13, wherein the yeast is selectedfrom the group consisting of Pichia, Candida, Torulopsis, Arxula,Hensenula, Yarrowia, Kluyveromyces, Saccharomyces, Komagataella,preferably a methylotrophic yeast.

15. Method according to definition 14, wherein the yeast is Pichiapastoris, Komagataella pastoris, K. phaffii, or K. pseudopastoris.

16. Method according to any of definitions 1 to 15, wherein the promoteris not natively associated with the nucleotide sequence encoding thePOI.

17. Method according to any of definitions 1 to 16, wherein the POI is aheterologous protein, preferably selected from therapeutic proteins,including antibodies or fragments thereof, enzymes and peptides, proteinantibiotics, toxin fusion proteins, carbohydrate-protein conjugates,structural proteins, regulatory proteins, vaccines and vaccine likeproteins or particles, process enzymes, growth factors, hormones andcytokines, or a metabolite of a POI.

18. Method according to any of definitions 1 to 17, wherein the POI is aeukaryotic protein, preferably a mammalian protein.

19. Method according to any of definitions 1 to 18, wherein the POI is amultimeric protein, preferably a dimer or tetramer.

20. Method according to any of definitions 1 to 19, wherein the POI isan antigen binding molecule such as an antibody, or a fragment thereof.

21. Method according to any of definitions 1 to 20, wherein afermentation product is manufactured using the POI, a metabolite or aderivative thereof.

22. Method for controlling the expression of a POI in a recombinanteukaryotic cell under the transcriptional control of a carbon sourceregulatable promoter having a transcription strength of at least 15% ascompared to the native pGAP promoter of the cell, wherein the expressionis induced under conditions limiting the carbon source.

23. Method of producing a POI in a recombinant eukaryotic cell under thetranscriptional control of a carbon source regulatable promoter, whereinsaid promoter has a transcription strength of at least 15% as comparedto the native pGAP promoter of the cell.

24. Method according to any of definitions 1 to 23, wherein theregulatable promoter comprises a nucleic acid sequence selected from thegroup consisting of

a) pG1 (SEQ ID 1), pG3 (SEQ ID 2), pG4 (SEQ ID 4), pG6 (SEQ ID 3), pG7(SEQ ID 5), or pG8 (SEQ ID 6);

b) a sequence having at least 60% homology to pG1 (SEQ ID 1), pG3 (SEQID 2), pG4 (SEQ ID 4), pG6 (SEQ ID 3), pG7 (SEQ ID 5), or pG8 (SEQ ID6);

c) a sequence which hybridizes under stringent conditions to pG1 (SEQ ID1), pG3 (SEQ ID 2), pG4 (SEQ ID 4), pG6 (SEQ ID 3), pG7 (SEQ ID 5), orpG8 (SEQ ID 6); and

d) a fragment or variant derived from a), b) or c),

wherein said promoter is a functionally active promoter, which is acarbon source regulatable promoter capable of expressing a POI in arecombinant eukaryotic cell at a transcription rate of at least 15% ascompared to the native pGAP promoter of the cell.

25. Method according to definition 24, wherein the variant of pG1 (SEQID 1), pG3 (SEQ ID 2), pG4 (SEQ ID 4), pG6 (SEQ ID 3), pG7 (SEQ ID 5),or pG8 (SEQ ID 6), is a functionally active variant selected from thegroup consisting of homologs with at least about 60% nucleotide sequenceidentity, homologs obtainable by modifying the parent nucleotidesequence by insertion, deletion or substitution of one or morenucleotides within the sequence or at either or both of the distal endsof the sequence, preferably comprising or consisting of a nucleotidesequence of at least 200 bp, and analogs derived from species other thanPichia pastoris.

26. Method according to definition 24 or 25, wherein the functionallyactive variant of pG1 is selected from the group consisting of pG1a (SEQID 41), pG1b (SEQ ID 42), pG1c (SEQ ID 43), pG1d (SEQ ID 44), pG1e (SEQID 45) and pG1f (SEQ ID 46).

27. An isolated nucleic acid comprising a nucleic acid sequence selectedfrom the group consisting of

a) pG1 (SEQ ID 1), pG3 (SEQ ID 2), pG6 (SEQ ID 3), pG7 (SEQ ID 5), orpG8 (SEQ ID 6);

b) a sequence having at least 60% homology to pG1 (SEQ ID 1), pG3 (SEQID 2), pG6 (SEQ ID 3), pG7 (SEQ ID 5), or pG8 (SEQ ID 6);

c) a sequence which hybridizes under stringent conditions to pG1 (SEQ ID1), pG3 (SEQ ID 2), pG6 (SEQ ID 3), pG7 (SEQ ID 5), or pG8 (SEQ ID 6);and

d) a fragment or variant derived from a), b) or c),

wherein said nucleic acid comprises a functionally active promoter,which is a carbon source regulatable promoter capable of expressing aPOI in a recombinant eukaryotic cell at a transcription rate of at least15% as compared to the native pGAP promoter of the cell.

28. Nucleic acid according to definition 27, wherein the variant of pG1(SEQ ID 1), pG3 (SEQ ID 2), pG6 (SEQ ID 3), pG7 (SEQ ID 5), or pG8 (SEQID 6) is a functionally active variant selected from the groupconsisting of homologs with at least about 60% nucleotide sequenceidentity, homologs obtainable by modifying the parent nucleotidesequence by insertion, deletion or substitution of one or morenucleotides within the sequence or at either or both of the distal endsof the sequence, preferably with a nucleotide sequence of at least 200bp, and analogs derived from species other than Pichia pastoris.

29. Nucleic acid according to definition 27 or 28, wherein thefunctionally active variant of pG1 is selected from the group consistingof pG1a (SEQ ID 41), pG1b (SEQ ID 42), pG1c (SEQ ID 43), pG1d (SEQ ID44), pG1e (SEQ ID 45) and pG1f (SEQ ID 46).

30. An expression construct comprising a nucleic acid according to anyof the definitions 27 to 29 operably linked to a nucleotide sequenceencoding a POI under the transcriptional control of said promoter, whichnucleic acid is not natively associated with the nucleotide sequenceencoding the POI.

31. Vector comprising the construct according to definition 30.

32. A recombinant eukaryotic cell comprising the construct of definition30, or the vector of definition 31.

33. A cell according to definition 31, which is selected from the groupconsisting of mammalian, insect, yeast, filamentous fungi and plant celllines, preferably a yeast.

34. A cell according to definition 32, wherein the yeast is selectedfrom the group consisting of Pichia, Candida, Torulopsis, Arxula,Hensenula, Yarrowia, Kluyveromyces, Saccharomyces, Komagataella,preferably a methylotrophic yeast.

35. A cell according to definition 34, wherein the yeast is Pichiapastoris, Komagataella pastoris, K. phaffii, or K. pseudopastoris.

36. A cell of any of definitions 32 to 35, which has a higher specificgrowth rate in the presence of a surplus of carbon source relative toconditions of limited carbon source.

37. Method to identify a carbon source regulatable promoter fromeukaryotic cells, comprising the steps of

a) cultivating eukaryotic cells in the presence of a carbon source in abatch culture under cell growing conditions,

b) further cultivating the cells in a fed batch culture in the presenceof a limited amount of a supplemental carbon source,

c) providing samples of the cell culture of step a) and b), and

d) performing transcription analysis in said samples to identify aregulatable promoter that shows a higher transcriptional strength incells of step b) than in cells of step a).

38. Method according to definition 37, wherein the transcriptionanalysis is quantitative or semi-quantitative, preferably employing DNAmicroarrays, RNA sequencing and transcriptome analysis.

Specific examples relate to fed-batch fermentation of a recombinantproduction P. pastoris cell line producing reporter proteins, employinga glycerol batch medium and a glucose fed batch medium. Comparativepromoter activity studies have proven that the promoter according to theinvention may be successfully activated to induce recombinant proteinproduction.

According to a further example, human serum albumin (HSA) was producedas a POI under the control of the glucose-limit induced promoters, andthe HSA yield and gene copy number determined.

According to another example, fed-batch cultivation of P. pastorisstrains expressing HSA under the control of a promoter according to theinvention was performed. Induction of the promoter activity underglucose-limiting conditions was found to be even more than 120 fold withpG1, and more than 20 fold with pG6, compared to the repressed state.

Further examples refer to expressing a porcine carboxypeptidase B asmodel protein under transcriptional control of pG1 and pG6 promoter.

Yet, a further example refers to the expression of an antibody fragmentunder the transcriptional control of pG1.

A further example proves the functional activity of variants of apromoter according to the invention, such as fragments of pG1 with alength in the range of 300 to 1000 bp. Additional experiments have shownthat even shorter fragments of pG1 were functionally active in a similarsetting, such as fragments ranging between 200 and 1000 bp, or fragmentsranging between 250 and 1000.

The foregoing description will be more fully understood with referenceto the following examples. Such examples are, however, merelyrepresentative of methods of practicing one or more embodiments of thepresent invention and should not be read as limiting the scope ofinvention.

EXAMPLES

Examples below illustrate the materials and methods used to identify newregulatable promoters and to analyze their expression properties inPichia pastoris.

Example 1: Identification of Strong, Efficiently Regulated Genes in P.pastoris in Glucose-Limited Conditions

In order to identify strong, efficiently regulated genes and theirrespective promoters of P. pastoris in glucose-limit conditions,analysis of gene expression patterns was done using microarrays. P.pastoris cells grown in a glycerol batch (surplus of carbon source) werecompared to cells which were cultivated in conditions where glucose wasgrowth limiting (chemostat), thereby simulating the course of a proteinproduction process, which is usually done in fed batch mode.

a) Strain

A wild type P. pastoris strain (CBS2612, CBS-KNAW Fungal BiodiversityCentre, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands),which can grow on minimal media without supplements, was used.

b) Cultivation of P. pastoris

Fermentations were performed with Minifors reactors (Infors-HT,Switzerland) with a final working volume of 2.5 L.

Following media were used:

PTM₁ Trace Salts Stock Solution Contained Per Liter

6.0 g CuSO₄.5H₂O, 0.08 g NaI, 3.36 g MnSO₄.H₂O, 0.2 g Na₂MoO₄.2H₂O, 0.02g H₃BO₃, 0.82 g CoCl₂, 20.0 g ZnCl₂, 65.0 g FeSO₄.7H₂O, 0.2 g biotin and5.0 ml H₂SO₄ (95%-98%).

Glycerol Batch Medium Contained Per Liter

2 g Citric acid monohydrate (C₆H₈O₇.H₂O), 39.2 g Glycerol, 20.8 gNH₄H₂PO₄, 0.5 g MgSO₄.7H₂O, 1.6 g KCl, 0.022 g CaCl₂.2H₂O, 0.8 mg biotinand 4.6 ml PTM1 trace salts stock solution. HCl was added to set the pHto 5.

Glycerol Fed-Batch Medium Contained Per Liter

632 g glycerol, 8 g MgSO₄.7H₂O, 22 g KCl, and 0.058 g CaCl₂.2H₂O.

Chemostat Medium Contained Per Liter

2 g Citric acid monohydrate (C₆H₈O₇.H₂O), 99.42 g glucose monohydrate,22 g NH₄H₂PO₄, 1.3 g MgSO₄.7H₂O, 3.4 g KCl, 0.02 g CaCl₂.2H₂O, 0.4 mgbiotin and 3.2 ml PTM1 trace salts stock solution. HCl was added to setthe pH to 5.

The dissolved oxygen was controlled at DO=20% with the stirrer speed(500-1250 rpm). Aeration rate was 60 L h⁻¹ air, the temperature wascontrolled at 25° C. and the pH setpoint of 5 was controlled withaddition of NH₄OH (25%).

To start the fermentation, 1.5 L batch medium was sterile filtered intothe fermenter and P. pastoris was inoculated (from an overnightpre-culture in YPG, 180 rpm, 28° C.) with a starting optical density(OD600) of 1. The batch phase of approximately 25 h reached a drybiomass concentration of approximately 20 g/L, it was followed by a 10 hexponential fed batch with glucose medium, leading to a dry biomassconcentration of approximately 50 g/L. Then, the volume was reduced to1.5 L and the chemostat cultivation was started with a feed/harvest rateof 0.15 L h⁻¹, resulting in a constant growth rate of μ=0.1. Thefermentation was terminated 50 h after the chemostat start.

This fermentation has been performed three times to obtain thebiological replicates necessary for reliable microarray analysis.

Carbon limited conditions (no detectable residual glucose) during thechemostat were verified by HPLC analysis of the culture supernatant.

c) Sampling

Samples were taken at the end of the glycerol batch phase and in steadystate conditions of the glucose chemostat. Routine sampling asdetermination of optical density or yeast dry mass, qualitativemicroscopic inspection and cell viability analysis was done alongsideduring each fermentation. For microarray analysis, samples were takenand treated as follows: For optimal quenching, 9 mL cell culture brothwas immediately mixed with 4.5 mL of ice cold 5% phenol (Sigma) solution(in Ethanol abs.), and aliquoted. Each 2 mL were centrifuged (13200 rpmfor 1 minute) in precooled collection tubes (GE healthcare, NJ),supernatant was removed completely and the tubes were stored at −80° C.until RNA purification.

d) RNA purification and sample preparation for microarray hybridization

The RNA was isolated using TRI reagent according to the suppliersinstructions (Ambion, US). The cell pellets were resuspended in TRIreagent and homogenized with glass beads using a FastPrep 24 (M.P.Biomedicals, CA) at 5 m s⁻¹ for 40 seconds. After addition ofchloroform, the samples were centrifuged and the total RNA wasprecipitated from the aqueous phase by adding isopropanol. The pelletwas washed with 70% ethanol, dried and re-suspended in RNAse free water.RNA concentrations were determined by measuring OD260 using a Nanodrop1000 spectrophotometer (Nano Drop products, DE). Remaining DNA from thesamples was removed using the DNA free Kit (Ambion, CA). Sample volumeequal to 10 μg RNA was diluted to 50 μL in RNAse free water, then DNAsebuffer I and rDNAse I were added and incubated at 37° C. for 30 minutes.After addition of DNAse Inactivation Reagent, the sample was centrifugedand the supernatant was transferred into a fresh tube. RNAconcentrations were determined again as described above. Additionally,RNA integrity was analyzed using RNA nano chips (Agilent). To monitorthe microarray workflow from amplification and labelling tohybridisation of the samples, the Spike In Kit (Agilent, Product Nr.:5188-5279) was used as positive control. It contains 10 differentpolyadenylated transcripts from an adenovirus, which are amplified,labelled and cohybridised together with the own RNA samples. The sampleswere labelled with Cy 3 and Cy 5 using the Quick Amp Labelling Kit(Agilent, Prod. No.: 5190-0444). Therefore 500 ng of purified sample RNAwere diluted in 8.3 μL RNAse free water, 2 μL Spike A or B, and 1.2 μLT7 promoter primer were added. The mixture was denatured for 10 minutesat 65° C. and kept on ice for 5 minutes. Then 8.5 μL cDNA mastermix (persample: 4 μL 5× first strand buffer, 2 μL 0.1 M DTT, 1 μL 10 mM dNTPmix, 1 μL MMLV-RT, 0.5 μL RNAse out) were added, incubated at 40° C. for2 hours, then transferred to 65° C. for 15 minutes and put on ice for 5minutes. The transcription mastermix (per sample: 15.3 μL nuclease freewater, 20 μL transcription buffer, 6 μL 0.1 M DTT, 6.4 μL 50% PEG, 0.5μL RNAse Inhibitor, 0.6 μL inorg. phosphatase, 0.8 μL T7 RNA Polymerase,2.4 μL Cyanin 3 or Cyanin 5) was prepared and added to each tube andincubated at 40° C. for 2 hours. In order to purify the obtainedlabelled cRNA, the RNeasy Mini Kit (Qiagen, Cat. No. 74104) was used.Samples were stored at −80° C. Quantification of the cRNA concentrationand labelling efficiency was done at the Nanodrop spectrophotometer.

e) Microarray Analysis

In order to identify strong, efficient regulated genes inglucose-limited chemostat cultivations, the three biological samplereplicates thereof were compared with the same reference and in onedyeswap each. The reference sample was generated by combining theglycerol batch cultivation samples in equal amounts.

The Gene Expression Hybridisation Kit (Agilent, Cat. No. 5188-5242) wasused for hybridisation of the labelled sample cRNAs. For the preparationof the hybridisation samples each 300 ng cRNA (Cy3 and Cy 5) and 6 μL10-fold blocking agent were diluted with nuclease free water to a finalvolume of 24 μL. After addition of 1 μL 25-fold fragmentation buffer,the mixture was incubated at 60° C. for 30 minutes. Then 25 μL GExHybridisation Buffer HI-RPM was added to stop the reaction. Aftercentrifugation for one minute with 13,200 rpm, the sample was chilled onice and used for hybridisation immediately. In-house designed P.pastoris specific oligonucleotide arrays (AMAD-ID: 026594, 8×15K customarrays, Agilent) were used. Microarray hybridisation was done accordingto the Microarray Hybridisation Chamber User Guide (Agilent G2534A).First, the gasket slide was uncovered and put onto the chamber base,Agilent label facing up. The sample (40 μL per array) was loaded in themiddle of each of the eight squares. Then the microarray slide wascarefully put onto the gasket slide (Agilent label facing down) and thechamber cover was placed on and fixed with the clamp. Hybridisation wasdone in the hybridisation oven for 17 hours at 65° C. Before scanning,the microarray chip was washed. Therefore, the chamber was dismantled,and the sandwich slides were detached from each other while submerged inwash buffer 1. The microarray was directly transferred into another dishwith wash buffer 1, washed for 1 minute, transferred into wash buffer 2(temperature at least 30° C.) and washed for another minute. Afterdrying of the microarray slide by touching the slide edge with a tissue,it was put into the slide holder (Agilent label facing up). The slideholder was put into the carousel and scanning was started.

f) Data Acquisition and Statistical Evaluation of Microarray Data

Images were scanned at a resolution of 50 nm with a G2565AA Microarrayscanner (Agilent) and were imported into the Agilent Feature Extraction9.5 software. Agilent Feature Extraction 9.5 was used for thequantification of the spot intensities. The raw mean spot intensity datawas then imported into the open source software R for furthernormalisation and data analysis.

For data preprocessing and normalization the R packages limma, vsn andmarray were used. The intensity data was not background corrected andnormalized with VSN, after normalization it was transformed into log 2ratios of the Cy5 channel against the Cy3 channel. Differentialexpression was calculated using the Imfit and eBayes function of thelimma package.

The microarray data was browsed for entries with both, high differencein expression level between repressed to induced state (fold change) aswell as high signal intensity in the induced state in order to identifystrongly expressed, efficiently regulated genes. A list of the selectedgenes is shown in Table 1, with the fold change meaning the signalintensity in the induced state divided by the signal intensity in therepressed state. The data of pGAP and pMLS1, pICL1 are added asreferences.

TABLE 1 Microarray data of the promoters selected for furthercharacterization and of pGAP, ICL1 and MLS1 as controls % of annota-intensity/ tion/ transcrip- yeast Fold- Inten- tion name homolog geneidentifier change sity* strength pGAP TDH3 PAS_chr2-1_0437 0.79 41052.5100.0 pG1 — PAS_chr1-3_0011 29.86 86312.9 210.2 pG3 YPR127WPAS_chr4_0550 2.66 15644.4 38.1 pG4 — PAS_chr4_0043 2.57 15664.8 38.2pG6 ALD4 PAS_chr2-1_0853 2.10 26888.4 65.5 MLS1 MLS1 PAS_chr4_0191 0.811446.9 3.5 ICL1 ICL1 PAS_chr1-4_0338 1.71 2574.3 6.3 pG7 HXT6PAS_chr1-4_0570 3.3 13336.5 32.5 pG8 SFL1 PAS_chr1-3_0165 2.1 9929.124.2 *of induced state in green channel

Example 2: Comparative Promoter Activity Studies of the Newly IdentifiedPromoters in P. pastoris Using eGFP as Intracellularly ExpressedReporter Gene

In order to analyze the properties of the newly identified promotersunder glucose limit conditions, shake flask screenings were performed asfollows: Pre-culture for 24 hours was done with rich medium containingglycerol as carbon source—simulating the batch phase of the process(repressed state of the promoters), which was followed by the mainculture with minimal medium and glucose feed beads—simulating theglucose-limited fed batch phase of the process (induced state of thepromoters).

a) Strain & Expression Vector

The P. pastoris wild type strain (CBS2612, CBS-KNAW Fungal BiodiversityCentre, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands)was used as host strain. Transformation of the strain was carried outwith an in-house vector named pPUZZLE (Stadlmayr et al. J. Biotechnol2010 December; 150(4):519-29), comprising of an origin of replicationfor E. coli (pUC19), an antibiotic resistance cassette (Sh ble geneconferring resistance to Zeocin) for selection in E. coli and yeast, anexpression cassette for the gene of interest (GOI) consisting of amultiple cloning site and the S. cerevisiae CYC1 transcriptionterminator, and a locus for integration into the P. pastoris genome (3.AOX1 region).

b) Amplification and Cloning of the Newly Identified Promoters pG1, pG3,pG4 and pG6 into pPUZZLE Expression Vector Containing eGFP as 001

A list of the newly identified promoter sequences and their respectivegenes (see Example 1) is shown in Table 2. 1000 bp of the 5′-non codingregion of the respective genes up to the start codon ATG were amplifiedby PCR (Phusion Polymerase, New England Biolabs) as promoter sequencesusing the primers shown in Table 2. These sequences were cloned into thepPUZZLE expression vector pPM1aZ10_eGFP, resulting in pPM1aZ10_pG1_eGFP,pPM1aZ10_pG3_eGFP, pPM1aZ10_pG4_eGFP and pPM1aZ10_pG6_eGFP.Additionally, the vector pPM1aZ10_pGAP_eGFP, containing the commonlyused promoter of glyceraldehyde 3-phosphate dehydrogenase promoter (pGAPof P. pastoris, here SEQ ID 25) was used as reference. The promoterswere inserted upstream of the start codon of the eGFP gene using theApaI and the SbfI restriction sites (see Tables 2 and 3). Thecorrectness of the promoter sequences was verified by Sanger sequencing.

TABLE 2 Primers for PCR amplification of the promoters Name TargetSequence T_(M) Restriction site pG1_fw pG1 SEQ ID 14 70.8 ApaIGATAGGGCCCCAAACATTTGCT CCCCCTAGTCTC pG1_back pG1 SEQ ID 15 69.8 SbfIGATACCTGCAGGAAGGGTGGAA TTTTAAGGATCTTTTAT pG3_fw pG3 SEQ ID 16 70.4 ApaIGATAGGGCCCCAGCAATCCAGT AACCTTTTCTGAAT pG3_back pG3 SEQ ID 17 70.2 SbfIGATACCTGCAGGTTGAGTTCAAT AAATTGTCCGGGA pG4_fw pG4 SEQ ID 18 70.4 ApaIGATAGGGCCCTGGACTGTTCAAT TTGAAGTCGATG pG4_back pG4 SEQ ID 19 70 SbfIGATACCTGCAGGGGATAAAGGTA AGGGAAAAAAGCAA pG6_fw pG6 SEQ ID 20 70.6 ApaIGATAGGGCCCAGACCAGCAGTTT AACTACGCAAATC pG6_back pG6 SEQ ID 21 70.7 SbfIGATACCTGCAGGCTTTTCTTTGGG CAAGGAAAAATC pG7_fw pG7 SEQ ID 22 69.1 ApaIGATAGGGCCCAATTGATTAAGTTCAGT GAAATTTCAAAC pG7_back pG7 SEQ ID 23 70.9SbfI GATACCTGCAGGATTATATTATGGGGA ATAATGAAGAGAAGG pG8_fw pG8 SEQ ID 2471.5 ApaI GATAGGGCCCCTGCACAACCATTGCC AGTAAGG pG8_back pG8 SEQ ID 25 70.4SbfI GATACCTGCAGGTTTTTAGAAGAGGG AGAACTTAGATTGG

TABLE 3 Amplification primers, cloning enzymes and the length of thecloned promoters Cloning enzyme Fragment promoter 5′primer 3′primer 5′3′ length pG1 pG1_fw pG1_back Apal Sbfl 988 pG3 pG3_fw pG3_back ApalSbfl 1011 pG4 pG4_fw pG4_back Apal Sbfl 1022 pG6 pG6_fw pG6_back ApalSbfl 1022 pG7 pG7_fw pG7_back Apal Sbfl 1022 pG8 pG8_fw pG8_back ApalSbfl 1022

c) Expression of eGFP in P. pastoris for Analysis of the PromoterActivity

All plasmids were linearized with AscI within the 3′AOX genomeintegration region prior to electroporation (2 kV, 4 ms, GenePulser,BioRad) into electrocompetent P. pastoris.

Selection of positive transformants was performed on YPD plates (perliter: 10 g yeast extract, 20 g peptone, 20 g glucose, 20 g agar-agar)plates containing 25 μg/mL of Zeocin (Invivogen, CA). Colony PCR wasused to ensure the presence of the transformed plasmid. Therefore,genomic DNA was gained by cooking and freezing of P. pastoris coloniesfor 5 minutes each and directly applied for PCR with the appropriateprimers. For expression screening, a single colony was inoculated inliquid YPG-Zeo medium (per liter: 20 g peptone, 10 g yeast extract, 12.6g glycerol and 25 mg Zeocin) as pre-culture. After approximately 24 hthe pre-culture was used to inoculate the main culture with an OD600 of0.1 in 10 ml YP medium (per liter: 20 g peptone, 10 g yeast extract) and2 glucose feed beads (Kuhner, CH). Glucose-limiting growth conditionswere achieved due to the slow glucose release kinetics of these feedbeads, which is described by the following equation:(Glucose)=1.63*t0.74 [mg/Disc]. Samples were taken at the end of thepre-culture, and 24 and 48 hours after inoculation of the main culture.Cell density was determined by measuring OD600, eGFP expression wasanalyzed by flow cytometry as described in Stadlmayr et al. (J.Biotechnology 2010 December; 150(4):519-29). For each sample 10,000cells were analyzed. Auto-fluorescence of P. pastoris was measured usinguntransformed P. pastoris wild type cells and subtracted from thesignal. Relative eGFP expression levels (fluorescence intensity relatedto cell size) are shown as percentage of eGFP expression level of aclone expressing eGFP under the control of the constitutive pGAPpromoter. Further similar studies are done with the promoters pG7 andpG8. Cloning is done as described in example 2b, except that the wildtype P. pastoris strain X-33 (Invitrogen) was used for thetransformation of pPM1aZ10_pG7_eGFP and pPM1aZ10_pG8_eGFP. Used primersand cloning fragments are listed in Tables 2 and 3. The results areshown in Table 4.

TABLE 4 Screening results of eGFP expressing P. pastoris clones underthe control of the novel promoters; Shown data (Fluorescence/cell size)is related to pGAP; pre-culture main culture batch end stdev 48 h stdevpG1 7.6 0.2 242.8 59.5 pG3 −5.1 2.4 25.4 5.5 pG4 −6.3 0.2 113.6 26.3 pG63.3 0.8 158.9 146.9 pG7 49.4 7.4 115.7 16.2 pG8 0.8 4.1 36.1 21.1

d) Determination of eGFP Gene Copy Numbers (GCN) in SelectedeGFPexpressing Clones

Expression strength is often correlated to the number of expressioncassettes integrated into the P. pastoris genome. Therefore the genecopy number of eGFP was determined. Genomic DNA was isolated using theDNeasy Blood&Tissue Kit (Quiagen, Cat. No. 69504). Gene copy numberswere determined using quantitative PCR. Therefore, SensiMix SYBR Kit(Bioline, QT605-05) was used. The Sensi Mix SYBR was mixed with theprimers and the sample and applied for real time analysis in a real-timePCR cycler (Rotor Gene, Qiagen). A list of the primers is shown in Table5. All samples were analyzed in tri- or quadruplicates. Rotor Genesoftware was used for data analysis. The actin gene ACT1 was used ascalibrator. Results are shown in Table 6.

TABLE 5 Primers for gene copy nuber determination by real-time PCRprimer target sequence T_(M) [° C.] product lengh PpACT1_Up ActSEQ ID 26 61.3 148 bp CCTGAGGCTTTGTTCC ACCCATCT PpACT1_Low Act SEQ ID 2761.4 148 bp GGAACATAGTAGTACC ACCGGACATAACGA PpeGFP_Up GFP SEQ ID 28 61.4124 bp TCGCCGACCACTACCA GCAGAA PpeGFP_Low GFP SEQ ID 29 61.6 124 bpACCATGTGATCGCGCT TCTCGTT

TABLE 6 Screening results (fluorescence/cell size related to pGAP) andgene copy numers of chosen P. pastoris clones expressing eGFP under thecontrol of pG1 and pG6; % of pGAP_eGFP fluorescence/size main cultureGCN pre culture 24 h 48 h pG1#8 1 7.32 33.00 184.30 pG1#9 1 7.73 33.96303.21 pG1#12 2 7.75 33.32 240.92 pG6#48 1 3.45 2.07 56.11 pG6#50 2 4.0023.18 327.14 pG6#53 1 2.52 9.78 93.51

e) Analysis of pG1 Promoter Strength in Fed-Batch Fermentation of OneeGFP Clone

Fed batch fermentations were performed in DASGIP reactors with a finalworking volume of 0.7 L.

Following media were used:

PTM₁ Trace Salts Stock Solution Contained Per Liter

6.0 g CuSO₄.5H₂O, 0.08 g NaI, 3.36 g MnSO₄.H₂O, 0.2 g Na₂MoO₄.2H₂O, 0.02g H₃BO₃, 0.82 g CoCl₂, 20.0 g ZnCl₂, 65.0 g FeSO₄.7H₂O, 0.2 g biotin and5.0 ml H₂SO₄ (95%-98%).

Glycerol Batch Medium Contained Per Liter

2 g Citric acid monohydrate (C₆H₈O₇.H₂O), 39.2 g Glycerol, 12.6 gNH₄H₂PO₄, 0.5 g MgSO₄.7H₂O, 0.9 g KCl, 0.022 g CaCl₂.2H₂O, 0.4 mg biotinand 4.6 ml PTM1 trace salts stock solution. HCl was added to set the pHto 5.

Glucose Fed Batch Medium Contained Per Liter

464 g glucose monohydrate, 5.2 g MgSO₄.7H2O, 8.4 g KCl, 0.28 gCaCl₂.2H₂O, 0.34 mg biotin and 10.1 mL PTM1 trace salts stock solution.

The dissolved oxygen was controlled at DO=20% with the stirrer speed(400-1200 rpm). Aeration rate was 24 Lh⁻¹ air, the temperature wascontrolled at 25° C. and the pH setpoint of 5 was controlled withaddition of NH₄OH (25%).

To start the fermentation, 400 mL batch medium was sterile filtered intothe fermenter and P. pastoris clone pG1_eGFP# 8 was inoculated (frompre-culture) with a starting optical density (OD600) of 1. The batchphase of approximately 25 h (reaching a dry biomass concentration ofapproximately 20 g/L) was followed by a glucose-limited fed batchstarting with an exponential feed for 7 h and a constant feed rate of 15g/L for 13 h, leading to a final dry biomass concentration ofapproximately 100 g/L. Samples were taken during batch and fed batchphase, and analyzed for eGFP expression using a plate reader (Infinite200, Tecan, CH). Therefore, samples were diluted to an optical density(OD600) of 5. Results are shown in Table 7 as relative fluorescence perbioreactor (FL/r).

TABLE 7 Relative fluorescence per bioreactor of two different P.pastoris clones expressing eGFP under the control of pGAP or pG1 in anoptimized fed batch fermentation. pGAP_eGFP#2 pG1_eGFP#8 t [h] FL/r t[h] FL/r −1.7 176.77 −0.38 131.95 0.0 166.52 0.00 108.76 0.5 199.59 0.28100.35 1.0 195.94 0.62 121.36 1.5 173.68 1.12 161.16 2.0 219.00 1.62162.69 3.0 321.14 2.12 148.34 7.0 494.60 3.12 205.20 19.1 1150.96 7.12373.08 20.0 1000.37 19.70 1745.65 21.12 1831.52

Example 3: Comparative Promoter Activity Studies of the Newly IdentifiedPromoters in P. pastoris Using Human Serum Albumin (HSA) asExtracellular Expressed Reporter Gene

In order to analyze the properties of the newly identified promotersunder glucose limit conditions, shake flask screenings were performed asfollows: Pre-culture for 24 hours was done with rich medium containingglycerol as carbon source—simulating the batch phase of the process(repressed state of the promoters), which was followed by the mainculture with minimal medium and glucose feed beads—simulating theglucose-limited fed batch phase of the process (induced state of thepromoters).

a) Strain & Expression Vector

The P. pastoris wild type strain (CBS2612, CBS-KNAW Fungal BiodiversityCentre, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands)was used as host strain. Transformation of the strain was carried outwith an in-house vector named pPUZZLE (Stadlmayr et al. J. Biotechnol2010 December; 150(4):519-29), selection of positive transformants wasbased on the Zeocin resistance. For secretory expression of human serumalbumin (HSA) its native secretion leader was used.

b) Amplification and Cloning of the Newly Identified Promoters pG1, pG3,pG4 and pG6 into an in-House Expression Vector

The four promoters amplified in Example 2b were cloned into the pPUZZLEexpression vector pPM1aZ10_HSA, resulting in pPM1aZ10_pG1_HSA,pPM1aZ10_pG3_HSA, pPM1aZ10_pG4_HSA and pPM1aZ10_pG6_HSA. Additionally,the vector pPM1aZ10_pGAP_HSA, containing the commonly used promoter ofglyceraldehyde 3-phosphate dehydrogenase promoter (pGAP) was used asreference. The promoters were inserted upstream of the start codon ofthe HSA gene using the ApaI and the SbfI restriction sites (see Table3). The correctness of the promoter sequences was verified by Sangersequencing.

c) Expression of HSA in P. pastoris Under Control of the NewlyIdentified Glucose-Limit Induced Promoters

All plasmids were linearized using AscI restriction enzyme prior toelectroporation (using a standard transformation protocol for P.pastoris) into P. pastoris. Selection of positive transformants wasperformed on YPD plates (per liter: 10 g yeast extract, 20 g peptone, 20g glucose, 20 g agar-agar) plates containing 25 μg/mL of Zeocin. ColonyPCR was used to ensure the presence of the transformed plasmid asdescribed in Example 2c.

For HSA expression screening, a single colony was inoculated in liquidYPG-Zeo medium (per liter: 20 g peptone, 10 g yeast extract, 12.6 gglycerol and 25 mg Zeocin) as pre-culture. After approximately 24 h thepre-culture was used to inoculate the main culture with an OD600 of 1 inYP medium (per liter: 20 g peptone, 10 g yeast extract) and glucose feedbeads (Kuhner, CH). Glucose-limiting growth conditions were achieved dueto the slow glucose release kinetics of these feed beads, which isdescribed by the following equation: (Glucose)=1.63*t^(0.74) [mg/Disc].Samples were taken at the end of the pre-culture, and 24 and 48 hoursafter inoculation of the main culture. Biomass concentration wasdetermined by measuring OD600 or wet cell weight. HSA concentration inthe culture supernatant was quantified by the Human Albumin ELISAQuantitation Set (Cat. No. E80-129, Bethyl Laboratories, Tex., USA)following the supplier's instruction manual. The HSA standard was usedwith a starting concentration of 400 ng mL⁻¹. Samples were dilutedaccordingly in sample diluent (50 mM Tris-HCl, 140 mM NaCl, 1% (w/v)BSA, 0.05% (v/v) Tween20, pH 8.0). HSA titers from screening of severalclones of each construct are presented in Table 8.

TABLE 8 Screening results of HSA expressing P. pastoris clones under thecontrol of pGAP, pG1 and pG6 HSA titer [mg L−1] clone pre culture mainculture 48 h pGAP_HSA #1 6.9 9.0 pGAP_HSA #2 9.0 8.6 pGAP_HSA #3 6.6 9.2pGAP_HSA #4 18.9  20.4  pGAP_HSA #5 9.6 8.3 pGAP_HSA #6 10.8  8.8pG1_HSA #19 0.6 6.9 pG1_HSA #20 0.6 6.7 pG1_HSA #21 0.1 7.0 pG1_HSA #22— — pG1_HSA #23 1.3 13.5  pG1_HSA #24 1.1 13.7  pG1_HSA #25 0.5 8.9pG1_HSA #26 0.5 9.2 pG1_HSA #27 0.6 7.3 pG1_HSA #28 0.6 6.1 pG1_HSA #290.6 6.4 pG1_HSA #30 0.6 7.1 pG6_HSA #31 0.3 1.8 pG6_HSA #32 0.3 1.7pG6_HSA #33 0.3 2.0 pG6_HSA #34 0.4 2.0 pG6_HSA #35 0.2 2.2 pG6_HSA #360.3 2.5 pG6_HSA #37 0.3 2.3 pG6_HSA #38 0.2 1.5 pG6_HSA #39 0.7 —pG6_HSA #40 0.2 2.4 pG6_HSA #41 0.4 — pG6_HSA #42 — 1.9

d) Determination of HSA Gene Copy Numbers

Genomic DNA isolation and qPCR measurement were performed as in Example2d, using the primers given in Table 9. Results are shown in Table 10.

TABLE 9 Primers for gene copy nuber determination by real-time PCRproduct primer target sequence length PpACT1_Up Act SEQ ID 30 148 bpCCTGAGGCTTTGTTCCACCCATCT PpACT1_Low Act SEQ ID 31 148 bpGGAACATAGTAGTACCACCGGACATAACGA PpHSA_Up HSA SEQ ID 32 135 bpAAACCTAGGAAAAGTGGGCAGCAAATGT PpHSA_Low HSA SEQ ID 33 135 bpACTCTGTCACTTACTGGCGTTTTCTCATG

TABLE 10 Screening and gene copy numer results of chosen P. pastorisclones expressing HSA under the control of pGAP, pG1 and pG6; HSA mgL⁻¹HSA mgL⁻¹ per GCN Clone GCN main culture main culture Mean STDEVpGAP_HSA#3 1 9.2 9.2 9.22 0.95 pGAP_HSA#4 2 20.4 10.2 pGAP_HSA#5 1 8.38.3 pG1_HSA#20 1 6.6 6.6 6.81 0.13 pG1_HSA#21 1 7.0 7.0 pG1_HSA#23 213.5 6.8 pG1_HSA#24 2 13.7 6.8 pG6_HSA#36 1 2.5 2.5 2.07 0.52 pG6_HSA#371 2.3 2.3 pG6_HSA#38 1 1.5 1.5

e) Fed-Batch Cultivation of P. pastoris Strains Expressing HSA UnderControl of the pG1 and pG6 Promoter

The fermentations were performed in DASGIP bioreactors with a finalworking volume of 0.7 L. The strain pG1_HSA# 23 had two HSA gene copies,the strain pG6_HSA# 36 carried only one HSA gene copy. Therefore twodifferent P. pastoris strains expressing HSA under control of pGAP(pGAP_HSA# 3 having one HSA gene copy, and pGAP_HSA# 4 having two HSAgene copies) were cultivated as reference. All fermentations wereperformed in duplicates.

Following media were used:

PTM₁ Trace Salts Stock Solution Contained Per Liter

6.0 g CuSO₄.5H₂O, 0.08 g NaI, 3.36 g MnSO₄.H₂O, 0.2 g Na₂MoO₄.2H₂O, 0.02g H₃BO₃, 0.82 g CoCl₂, 20.0 g ZnCl₂, 65.0 g FeSO₄.7H₂O, 0.2 g biotin and5.0 ml H₂SO₄ (95%-98%).

Glycerol Batch Medium Contained Per Liter 39.2 g Glycerol, 27.9 g H₃PO₄(85%), 7.8 g MgSO₄.7H₂O, 2.6 g KOH, 9.5 g K₂SO₄, 0.6 g CaSO₄.2H2O, 0.4mg biotin and 4.6 ml PTM1 trace salts stock solution. The pH wasadjusted to 5.85 after sterile filtering into the fermenter.

Glucose Fed Batch Medium Contained Per Liter

550 g glucose monohydrate, 6.5 g MgSO₄.7H₂O, 10 g KCl, 0.35 gCaCl₂.2H₂O, 0.4 mg biotin and 12 ml PTM1 trace salts stock solution.

The dissolved oxygen was controlled at DO=20% with the stirrer speed(400-1200 rpm). Aeration rate was 24 l h⁻¹ air, the temperature wascontrolled at 25° C. and the pH setpoint of 5.85 was controlled withaddition of NH₄OH (25%).

To start the fermentation, 400 ml batch medium was sterile filtered intothe fermenter and P. pastoris was inoculated (from pre-culture) with astarting optical density (OD600) of 1. The batch phase of approximately25 h reached a dry biomass concentration of approximately 20 g/L and itwas followed by a constant fed batch (for 100 hours) with glucosemedium, leading to a dry biomass concentration of approximately 100 g/L.The pH was 5.85 during batch, and kept at 5.85 throughout thefermentation. Samples were taken during batch and fed batch phase. HSAconcentration was quantified using the Human Albumin ELISA QuantitationSet (Bethyl, Cat. No. E80-129) as described in Example 3c. Biomassconcentration and HSA titers are shown in Table 11, the product yield(amount of HSA secreted per biomass, HSA/YDM) at the end of the batch(repressing conditions for pG1 and pG6) and the end of the fed batch(inducing conditions for pG1 and pG6) are given in Table 12. Thereby theinduction strategy could be verified. pG1 and pG6 are repressed undercarbon source surplus (in glycerol batch), showing nearly no detectableHSA in contrast to the pGAP driven clones. Induction of pG1 and pG6occurred upon the switch to C-limited conditions with the start of thefed batch phase. Induction of pG1 (HSA/YDM) was more than 120-foldcompared to the repressed state, induction of pG6 was more than 20-foldcompared to the repressed state, while nearly no change was observed forpGAP (3-fold increase in HSA/YDM compared to batch phase).

TABLE 11 Yeast dry mass and HSA titers at batch end and fed batch end of7 fermentations of P. pastoris clones expressing HSA under the controlof pGAP, pG1 or pG6. Batch end Fed batch end Fermen- HSA HSA tation timeYDM titer time YDM titer CLONE # [h] [gL⁻¹] [mgL⁻¹] [h] [gL⁻¹] [mgL⁻¹]pG1#23 A041 −1.1 24.7 0.5 99.6 125.3 328.6 pG1#23 A048 −0.3 23.9 0.5108.4 128.6 277.6 pG6#36 A045 −0.1 23.5 0.3 104.7 125.2 21.8 pG6#36 A049−0.3 24.4 0.3 108.4 129.0 26.9 pGAP#4 A044 −0.1 23.6 11.2 104.7 129.1141.4 pGAP#4 A051 −0.9 24.1 9.0 96.9 118.2 114.9 pGAP#3 A050 −0.9 24.25.0 96.9 117.7 57.8

TABLE 12 HSA titer per yeast dry mass at batch end and fed batch end of7 fermentations of P. pastoris clones expressing HSA under the controlof pGAP, pG1 or pG6. end of batch end of fed batch GCN mean mean foldHSA HSA/YDM SD HSA/YDM SD induction pG1#23 2 0.02 0.00 2.39 0.33 121.06pG6#36 1 0.01 0.00 0.19 0.02 21.39 pGAP#4 2 0.42 0.07 1.03 0.09 3.16pGAP#3 1 0.21 0.49

Example 4: Comparative Promoter Activity Studies in Various GlucoseConcentrations of the Newly Identified Promoters in P. pastoris UsingeGFP as Intracellular Expressed Reporter Gene

In order to analyze the properties of the newly identified promoters invarious glucose concentrations, shake flask screenings were performed asfollows: Pre-culture for 24 hours was done with rich medium containingglycerol as carbon source (repressed state of the promoters), which wasfollowed by the main culture with minimal medium and glucose as carbonsource (induced state of the promoters);

a) Strain & Expression Vector

The P. pastoris wild type strain (CBS2612, CBS-KNAW Fungal BiodiversityCentre, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands)is used as host strain. Transformation of the strain was carried outwith an in-house vector named pPUZZLE (Stadlmayr et al. J. Biotechnol2010 December; 150(4):519-29), selection of positive transformants wasbased on the Zeocin resistance.

b) Amplification and Cloning of the Newly Identified Promoters pG1, pG3,pG4 and pG6 into pPUZZLE Expression Vector Containing eGFP as GOI

Amplification and cloning is done as described in example 2.

c) Expression of eGFP in P. pastoris for Analysis of the PromoterActivity

Transformation and clone selection is done as described in Example 2.

For expression screening, a single colony is inoculated in liquidYPG-Zeo medium (per liter: 20 g peptone, 10 g yeast extract, 12.6 gglycerol and 25 mg Zeocin) as pre-culture. After approximately 24 h thepre-culture is used to inoculate the main culture with an OD600 of 0.01in 10 ml YP medium (per liter: 20 g peptone, 10 g yeast extract) andglucose as carbon source. Glucose is used in various concentrations from20 to 0.001 g L⁻¹.

Samples are taken after 1-8 hours after inoculation of the main culture.eGFP expression is analyzed by flow cytometry as described in Stadlmayret al. (J. Biotechnology 2010 December; 150(4):519-29), selection ofpositive transformants is based on the Zeocin resistance. For eachsample 10,000 cells are analyzed. Auto-fluorescence of P. pastoris ismeasured using untransformed P. pastoris wild type cells.

Example 5: Comparative Promoter Activity Studies of the Newly IdentifiedPromoters in P. pastoris Using Porcine Carboxypeptidase B (CpB) asExtracellular Expressed Reporter Gene

In order to analyze the properties of the newly identified promotersunder glucose limit conditions, shake flask screenings is performed asfollows: Pre-culture for 24 hours is done with rich medium containingglycerol as carbon source—simulating the batch phase of the process(repressed state of the promoters), which is followed by the mainculture with minimal medium and glucose feed beads—simulating theglucose-limited fed batch phase of the process (induced state of thepromoters);

a) Strain & Expression Vector

The P. pastoris wild type strain (CBS2612, CBS-KNAW Fungal BiodiversityCentre, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands)is used as host strain. Transformation of the strain is carried out withan in-house vector named pPUZZLE (Stadlmayr et al. J. Biotechnol 2010December; 150(4):519-29), selection of positive transformants is basedon the Zeocin resistance. For secretory expression of porcinecarboxypeptidase B (CpB) yeast alpha mating factor leader is used.

b) Amplification and Cloning of the Newly Identified Promoters pG1, pG3,pG4 and pG6 into an in-House Expression Vector

Two promoters amplified in Example 2b are cloned into the pPUZZLEexpression vector pPM1aZ30_aMF_CpB, resulting in pPM1aZ30_pG1_aMF_CpBand pPM1aZ30_pG6_aMF_CpB. Additionally, the vector pPM1dZ30_pGAP_CPB,containing the commonly used promoter of glyceraldehyde 3-phosphatedehydrogenase promoter (pGAP) is used as reference. The promoters areinserted upstream of the start codon of the CpB gene using the ApaI andthe SbfI restriction sites The correctness of the promoter sequences isverified by Sanger sequencing.

c) Expression of CpB in P. pastoris Under Control of the NewlyIdentified Glucose-Limit Induced Promoters

Plasmids are linearized using SpeI or SapI restriction enzyme prior toelectroporation (using a standard transformation protocol for P.pastoris) into P. pastoris. Selection of positive transformants isperformed on YPD plates (per liter: 10 g yeast extract, 20 g peptone, 20g glucose, 20 g agar-agar) plates containing 25 μg/mL of Zeocin. ColonyPCR is used to ensure the presence of the transformed plasmid asdescribed in Example 2c.

For CpB expression screening, a single colony is inoculated in liquidYPG-Zeo medium (per liter: 20 g peptone, 10 g yeast extract, 12.6 gglycerol and 25 mg Zeocin) as pre-culture. After approximately 24 h thepre-culture is used to inoculate the main culture with an OD600 of 1 inYP medium (per liter: 20 g peptone, 10 g yeast extract) and glucose feedbeads (Kuhner, CH). Glucose-limiting growth conditions are achieved dueto the slow glucose release kinetics of these feed beads, which isdescribed by the following equation: (Glucose)=1.63*t^(0.74) [mg/Disc].Samples are taken at the end of the pre-culture, and 24 and 48 hoursafter inoculation of the main culture. Biomass concentration isdetermined by measuring OD600 or wet cell weight. CpB concentration inthe culture supernatant is quantified by an enzymatic assay, based onthe conversion of hippuryl-L-arginine to hippuric acid by the CpB.Reaction kinetics are measured by monitoring the absorption at 254 nm at25° C. using a Hitachi U-2910 Spectrophotometer when the reaction isstarted. Samples and standards are buffered with assay buffer (25 mMTris, 100 mM HCl, pH 7.65) and are activated using activation buffer(0.01 mgL-1 Trypsin, 300 mM Tris, 1 μM ZnCl₂, pH 7.65). Activationbuffer without trypsin is used instead of sample as negative control.The reaction is started by adding the substrate solution (1 mMhippuryl-L-arginine in assay buffer).

d) Fed-Batch Cultivation of P. pastoris Strains Expressing CpB UnderControl of the pG6 Promoter

Fed batch fermentation is done as described in example 3e. The clonepPM1aZ10_pG6_CpB# 4 produced no detectable CpB in the batch and morethan 210 mg/L CpB at the end of the fed batch.

Example 6: Comparative Promoter Activity Studies of the Newly IdentifiedPromoters pG1 and pG6 in P. pastoris Multicopy Clones Using Human SerumAlbumin (HSA) as Extracellular Expressed Reporter Gene

In order to analyze the properties of the newly identified promotersunder glucose limit conditions, shake flask screenings are performed asfollows: Pre-culture for 24 hours is done with rich medium containingglycerol as carbon source—simulating the batch phase of the process(repressed state of the promoters), which is followed by the mainculture with minimal medium and glucose feed beads—simulating theglucose-limited fed batch phase of the process (induced state of thepromoters);

a) Strain & Expression Vector

The P. pastoris wild type strain (CBS2612, CBS-KNAW Fungal BiodiversityCentre, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands)is used as host strain. Transformation of the strain is carried out withan in-house vector named pPUZZLE (Stadlmayr et al. J. Biotechnol 2010December; 150(4):519-29), selection of positive transformants is basedon the Zeocin resistance. For secretory expression of human serumalbumin (HSA) its native secretion leader is used.

b) Amplification and Cloning of the Newly Identified Promoters pG1 andpG6 into an in-House Expression Vector

Two promoters amplified in Example 2b are cloned into the pPUZZLEexpression vector pPM1nZ30_HSA, resulting in pPM1nZ30_pG1_HSA and pPM1nZ30_pG6_HSA.

The promoters are inserted upstream of the start codon of the HSA geneusing the ApaI and the SbfI restriction sites. The correctness of thepromoter sequences is verified by Sanger sequencing.

c) Expression of HSA in P. pastoris Under Control of the NewlyIdentified Glucose-Limit Induced Promoters

All plasmids are linearized using AscI restriction enzyme prior toelectroporation (using a standard transformation protocol for P.pastoris) into P. pastoris. Selection of positive transformants isperformed on YPD plates (per liter: 10 g yeast extract, 20 g peptone, 20g glucose, 20 g agar-agar) plates containing 25 μg/mL of Zeocin. Genecopy number amplification is done as described in Marx et al. (FEMSYeast Res. 2009 December; 9(8):1260-70). Colony PCR is used to ensurethe presence of the transformed plasmid as described in Example 2c.

For HSA expression screening, a single colony is inoculated in liquidYPG-Zeo medium (per liter: 20 g peptone, 10 g yeast extract, 12.6 gglycerol and 25 mg Zeocin) as pre-culture. After approximately 24 h thepre-culture is used to inoculate the main culture with an OD600 of 1 inYP medium (per liter: 20 g peptone, 10 g yeast extract) and glucose feedbeads (Kuhner, CH). Glucose-limiting growth conditions are achieved dueto the slow glucose release kinetics of these feed beads, which isdescribed by the following equation: (Glucose)=1.63*t0.74 [mg/Disc].Samples are taken at the end of the pre-culture, and 24 and 48 hoursafter inoculation of the main culture. Biomass concentration isdetermined by measuring OD600 or wet cell weight. HSA concentration inthe culture supernatant is quantified by the Human Albumin ELISAQuantitation Set (Cat. No. E80-129, Bethyl Laboratories, Tex., USA)following the supplier's instruction manual. The HSA standard is usedwith a starting concentration of 400 ng mL⁻¹. Samples are dilutedaccordingly in sample diluent (50 mM Tris-HCl, 140 mM NaCl, 1% (w/v)BSA, 0.05% (v/v) Tween20, pH 8.0). HSA titers from a screening ofseveral multicopy clones and single copy clones from Example 3c arepresented in Table 13.

TABLE 13 Screening results of P. pastoris multicopy clones expressingHSA under the control of pGAP, pG1 and pG6 clone HSA titer (mg/L)pPM1aZ10_pG1_HSA#23 8.20 pPM1nZ30_pG1_HSA#C2 19.55pPM1nZ30_pG1_HSA#4*1000 21.59 pPM1nZ30_pG1_HSA#5*1000 21.33pPM1nZ30_pG1_HSA#X4 27.22 pPM1nZ30_pG1_HSA#X5 6.90 pPM1aZ10_pG6_HSA#361.55 pPM1nZ30_pG6_HSA#C6 14.12 pPM1nZ30_pG6_HSA#2*1000 15.85pPM1nZ30_pG6_HSA#X5 11.52 pPM1nZ30_pG6_HSA#X8 7.87

d) Determination of HSA Gene Copy Numbers

Genomic DNA isolation and qPCR measurement are performed as in Example2d, using the primers given in Table 9. Results are shown in Table 14.

TABLE 14 Screening and gene copy numer results of chosen P. pastorismulticopy clones expressing HSA under the control of pGAP, pG1 and pG6HSA mgL⁻¹ HSA mgL⁻¹ per GCN clone GCN main culture main culturepPM1nZ30_pG1_HSA#4*1000 11 21.59 1.89 pPM1nZ30_pG1_HSA#X4 17 27.22 1.64pPM1nZ30_pG6_HSA#C6 12 14.12 1.23 pPM1nZ30_pG6_HSA#2*1000 6 15.85 2.50e) Fed-Batch Cultivation of Multicopy P. pastoris Strains Expressing HSAUnder Control of the pG1 and pG6 Promoter

Fed batch fermentations are done as described in example 3e. The clonespPM1nZ30_pG1_HSA# 4*1000 and pPM1nZ30_pG6_HSA# C6 reached 1060 and 728mg/L HSA at the end of the fed batch, respectively.

Example 7: Comparative Promoter Activity Studies of the Newly IdentifiedPromoter pG1 in P. pastoris Using Antibody Fragment (Fab) asExtracellular Expressed Reporter Gene

In order to analyze the properties of the newly identified promotersunder glucose limit conditions, shake flask screenings is performed asfollows: Pre-culture for 24 hours is done with rich medium containingglycerol as carbon source—simulating the batch phase of the process(repressed state of the promoters), which is followed by the mainculture with minimal medium and glucose feed beads—simulating theglucose-limited fed batch phase of the process (induced state of thepromoters);

a) Strain & Expression Vector

The P. pastoris wild type strain (CBS2612, CBS-KNAW Fungal BiodiversityCentre, Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands)is used as host strain. Transformation of the strain is carried out withan in-house vector named pPUZZLE (Stadlmayr et al. J. Biotechnol 2010December; 150(4):519-29), selection of positive transformants is basedon the Zeocin resistance. For secretory expression of an antibody Fabfragment, yeast alpha mating factor leader is used.

b) Amplification and Cloning of the Newly Identified Promoter pG1 intoan in-House Expression Vector

The pG1 promoter amplified in Example 2b is cloned into the pPUZZLEexpression vector containing Fab as GOI as described in example 5b. Thepromoter is inserted upstream of the start codon of the Fab gene usingthe ApaI and the SbfI restriction sites. The correctness of the promotersequence is verified by Sanger sequencing.

c) Expression of Fab in P. pastoris Under Control of the NewlyIdentified Glucose-Limit Induced Promoter pG1

Plasmids are linearized using SpeI or SapI restriction enzyme prior toelectroporation (using a standard transformation protocol for P.pastoris) into P. pastoris. Selection of positive transformants isperformed on YPD plates (per liter: 10 g yeast extract, 20 g peptone, 20g glucose, 20 g agar-agar) plates containing 25 μg/mL of Zeocin. ColonyPCR is used to ensure the presence of the transformed plasmid asdescribed in Example 2c.

For Fab expression screening, a single colony is inoculated in liquidYPG-Zeo medium (per liter: 20 g peptone, 10 g yeast extract, 12.6 gglycerol and 25 mg Zeocin) as pre-culture. After approximately 24 h thepre-culture is used to inoculate the main culture with an OD600 of 1 inYP medium (per liter: 20 g peptone, 10 g yeast extract) and glucose feedbeads (Kuhner, CH). Glucose-limiting growth conditions are achieved dueto the slow glucose release kinetics of these feed beads, which isdescribed by the following equation: (Glucose)=1.63*t^(0.74) [mg/Disc].Samples are taken at the end of the pre-culture, and 24 and 48 hoursafter inoculation of the main culture. Biomass concentration isdetermined by measuring OD600 or wet cell weight. Fab expression levelsare quantified by ELISA using Anti-Human Kappa Light Chains (Bound andFree)-Alkaline Phosphatase antibody produced in goat. Fab titers from ascreening of several Fab expressing clones under the control of pGAP andpG1 are presented in Table 15.

TABLE 15 Screening results of P. pastoris clones expressing Fab underthe control of pGAP and pG1 Fab (mg/L) pPM1dZ30_pGAP_Fab#2 0.00pPM1dZ30_pGAP_Fab#5 0.70 pPM1dZ30_pGAP_Fab#7 0.68 pPM1aZ30_pG1_Fab#22.02 pPM1aZ30_pG1_Fab#3 0.70 pPM1aZ30_pG1_Fab#4 1.10 pPM1aZ30_pG1_Fab#50.00 pPM1aZ30_pG1_Fab#6 0.56 pPM1aZ30_pG1_Fab#9 0.66 pPM1aZ30_pG1_Fab#101.80 pPM1aZ30_pG1_Fab#11 1.64 pPM1aZ30_pG1_Fab#12 2.31pPM1aZ30_pG1_Fab#13 2.35 pPM1aZ30_pG1_Fab#14 2.27 pPM1aZ30_pG1_Fab#151.60 pPM1aZ30_pG1_Fab#16 1.45 pPM1aZ30_pG1_Fab#B9 2.89pPM1aZ30_pG1_Fab#B10 2.32 pPM1aZ30_pG1_Fab#B11 6.45 pPM1aZ30_pG1_Fab#B123.24 pPM1aZ30_pG1_Fab#B13 2.57 pPM1aZ30_pG1_Fab#B14 3.14pPM1aZ30_pG1_Fab#B15 3.23 pPM1aZ30_pG1_Fab#B16 2.61 pPM1aZ30_pG1_Fab#C110.58 pPM1aZ30_pG1_Fab#C2 1.46 pPM1aZ30_pG1_Fab#C3 12.38pPM1aZ30_pG1_Fab#C4 9.91 pPM1aZ30_pG1_Fab#C5 1.96 pPM1aZ30_pG1_Fab#C62.87 pPM1aZ30_pG1_Fab#C7 7.03 pPM1aZ30_pG1_Fab#C8 6.37

d) Fed-Batch Cultivation of P. pastoris Strains Expressing Fab UnderControl of the pG1 Promoter.

-   -   Fed batch fermentations are done similar as described in example        3e, but glucose fed batch as described in example 2e is used.        The clones pPM1aZ30_pG1_Fab# C4 and pPM1aZ30_pG1_Fab# C7 reached        165 and 131 mg/L Fab at the end of the fed batch, respectively.

Example 8: Exponential Fed-Batch Fermentation to Control the SpecificGrowth Rate at the Maximal Volumetric Productivity of the NewlyIdentified Promoters

Chemostat cultivations of P. pastoris clones expressing a reporter geneunder the control of the newly identified promoters are used todetermine the specific and volumetric productivity at different growthrates. As described by Maurer et al. (Microb Cell Fact. 2006 Dec. 11;5:37), exponential fed-batch fermentations can be used to grow a P.pastoris clone at a certain growth rate for improved production duringthe whole feed phase. Thereby the space-time yield can be optimized. Anoptimized feed was applied and the space-time yield of the fed batchphase was improved by more than 35%.

Example 9: Determination of Promoter/Transcription Strength: ComparativePromoter Activity Study to Identify Promoter Regulation on DifferentGlucose Concentrations Using eGFP as Intracellular Expressed ReporterGene

Regulation properties of a promoter are analyzed by screening clonesexpressing eGFP under the control of said promoter. Therefore, a singlecolony is inoculated in liquid YPG-Zeo medium (per liter: 20 g peptone,10 g yeast extract, 12.6 g glycerol and 25 mg Zeocin) as pre-culture.After approximately 24 h the pre-culture is used to inoculate the mainculture with an OD600 of 0.01 in 10 ml YP medium (per liter: 20 gpeptone, 10 g yeast extract) and glucose in different concentrations(20, 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.078, 0.039, 0.020, 0.010,0.005 and 0.002 g/L). A sample is taken after 6 hours and analyzed byflow cytometry as described by Stadlmayr et al. (J Biotechnol. 2010December; 150(4):519-29). Fluorescence related to cell size (forwardscatter to the power of 1.5) is calculated for each cell/data point andthe geometric mean thereof is used to compare eGFP expression levelsproduced in different glucose concentrations. A clone expressing eGFPunder the control of pGAP is used as reference (pGAP of P. pastoris,here SEQ ID 25). Auto-fluorescence of P. pastoris is measured usinguntransformed P. pastoris wild type cells and subtracted from thesignal. Table 16 shows the full induction of pG1 promoter at about 40mg/L glucose or less, and its transcription strength as compared to thenative pGAP promoter.

TABLE 16 Relative eGFP expression (related to pGAP) of a P. pastorisclone expressing eGFP under the control of the pG1 promoter in differentglucose concentrations (20-0.002 g/L) % of pGAP Glucose (g/L) 14.7 2017.4 10 23.7 5 25.4 2.5 28.2 1.25 30.6 0.625 36.9 0.3125 44.5 0.1562550.9 0.078125 56.2 0.0390625 55.0 0.0195313 57.5 0.0097656 59.20.0048828 59.6 0.0024414

Further similar studies were made to compare the relative transcriptionstrength of the de-repressed promoters pG1, pG3, pG4, pG6 and pG7. Aclone expressing eGFP under the control of one of the promoters wascultivated in YPG (20 g/L glycerol, repressed state) and then inoculatedin YP medium containing different amounts of glucose (20 to 0.002 g/L(D20, D10, . . . D0.002), induced state) and cultivated for 5-6 hours.Cells were analyzed by flow cytometry and results were evaluated asfollows: The fluorescence was related to cell size (forward scatter tothe power of 1.5) for each cell and the geometric mean thereof was usedfor comparison of different glucose concentrations. The concludingresult of these screenings are shown in FIG. 14, a diagram showing thelogarithmic glucose concentrations against relative fluorescence, givinga good picture of the induction behaviour of glucose-limit regulatablepromoters. FIG. 14 shows the full induction of pG1 promoter at about 40mg/L glucose or less, and of the promoters pG3, pG4 and pG6 at about 4g/L or less, and the transcription strength as compared to the nativepGAP promoter. The induction behaviour of pG7 is similar to pG1 (Datanot shown). Based on the previous results with pG8 it is assumed thatits induction behavior is in the range of the other promoters.

Example 10: Comparison of Prior Art pICL1 and pMLS1 Promoters to pG1 inthe Glucose Concentration Screening Assay

The comparative promoter activity study is performed according toExample 9, employing the pICL1 and pMLS1 promoters as a reference tocompare with the pG1 promoter according to the invention.

The activity of both pICL1 and pMLS1 promoters is found to be very weak,with no significant difference at high (D20: 20 g/L/Repression) or low(D0.04: 0.04 g/L Induction=De-Repression) glucose concentration. In anycase the activity is far less than the activity of the repressed pG1promoter in the same setting. Results are shown in Table 17 as promoteractivity in % relative to the pGAP promoter.

TABLE 17 Relative fluorescence of strains expressing eGFP under controlof pG1, pICL1 and pMLS1, respectively, grown either in medium containing20 g/L glucose (D20) or 0.04 g/L (D0.04). D20/Repression D0.04/InductionpG1#8 9.95 +/− 2.60 48.41 +/− 2.76  pICL1 2.68 +/− 1.78 5.07 +/− 0.90pMLS1 −1.26 +/− 0.54  0.58 +/− 0.22

Example 11: Comparison of Variants of pG1

Shorter variants of the pG1 promoter are cloned as described in example2a and screened similar as described in example 2c, but in a downscaledsetup using 24-well plates (Whatman, UK, Art. Nr. 7701-5110) andquarters of feed beads (12 mm, Kuhner, CH) instead of total ones. Clonesexpressing under the control of pG1 and pGAP are used as controls.Forward primers and lengths of pG1 and its variants are listed in Table18. There was no significant difference in the relative fluorescence ofcells expressing eGFP under the control of pG1 and the pG1 variants a-f.

TABLE 18 pG1 and its variants: forward primers and 5′ start and 3′endpositions in the pG1 sequence (SEQ ID 1). Sequences of pG1a-f seeFIG. 15 (SEQ ID 41-46). Length promoter primer 5′ 3′ (bp) pG1GATAGGGCCCCAAACATTTGCTCCCCCTAGTCTC  36 1001 988 SEQ ID 34 pG1 aGATAGGGCCCGGAATCTGTATTGTTAGAAAGAACGAG 143 1001 881 AG SEQ ID 35 pG1 bGATAGGGCCCCCATATTCAGTAGGTGTTTCTTGCAC 338 1001 686 SEQ ID 36 pG1 cGATAGGGCCCCTGCAGATAGACTTCAAGATCTCAGG 509 1001 515 SEQ ID 37 pG1 dGATAGGGCCCGACCCCGTTTTCGTGACAAATT 632 1001 392 SEQ ID 38 pG1 eGATAGGGCCCCCGGATAAGAGAATTTTGTTTGATTAT 674 1001 350 SEQ ID 39 pG1 fGATAGGGCCCGCCTGCTCCATATTTTTCCGG 719 1001 305 SEQ ID 40

The invention claimed is:
 1. A method of producing a promoter variantwhich is a variant of a promoter selected from the group consisting ofpG1 consisting of the nucleotide sequence of SEQ ID NO: 1, pG3consisting of the nucleotide sequence of SEQ ID NO: 2, pG4 consisting ofthe nucleotide sequence of SEQ ID NO: 4, pG6 consisting of thenucleotide sequence of SEQ ID NO: 3, pG7 consisting of the nucleotidesequence of SEQ ID NO: 5, and pG8 consisting of the nucleotide sequenceof SEQ ID NO: 6, which method comprises a) mutagenesis of the respectivepromoter nucleotide sequence thereby producing variants comprising anucleotide sequence of at least 250 bp and having at least 80% sequenceidentity to the respective promoter nucleotide sequence, b) determiningthe transcription rate of the variants, and c) selecting a promotervariant from said variants which is capable of producing a protein ofinterest (POI) in a recombinant eukaryotic cell by expressing anucleotide sequence encoding said POI under the transcriptional controlof said promoter variant, at a transcription rate of at least 20% ascompared to the pGAP promoter (SEQ ID NO:13).
 2. The method of claim 1,wherein in step b) the transcription rate of the variants is determinedwhen repressed and de-repressed to select a carbon source regulatablepromoter variant.
 3. The method of claim 1, wherein the promoter variantis selected which is regulatable by a carbon source such that i) it isrepressed in a cell when cultivating the cell with a basal carbon sourcerepressing the promoter, wherein the basal carbon source is a carbonsource suitable for cell growth; and ii) it is de-repressed and fullyinduced in the cell when cultivating the cell in the presence of 0-1 g/Lof a supplemental carbon source.
 4. The method of claim 3, wherein a)the basal carbon source is selected from the group consisting ofglucose, glycerol, ethanol, fructose, galactose, mannose, adisaccharide, and mixtures thereof; and/or b) wherein the supplementalcarbon source is selected from the group consisting of a hexose, adisaccharide, an alcohol, and mixtures thereof.
 5. The method of claim1, wherein the promoter variant is selected which is de-repressed andfully induced when cultivating the cell using a feed medium which ischemically defined and methanol-free.
 6. The method of claim 1, whereinthe transcription rate is determined in a cell culture employing a feedmedium that provides for a limited amount of the supplemental carbonsource, or an amount of 1 g/L or less of the supplemental carbon sourcein the culture medium.
 7. The method of claim 1, wherein in step a) saidmutagenesis includes modifying the respective promoter nucleotidesequence by insertion, deletion or substitution of one or morenucleotides within the sequence or at either or both of the distal endsof the sequence.
 8. The method of claim 1, wherein in step a) saidmutagenesis includes producing fragments of any of the pG1, pG3, pG4,pG7, or pG8 promoter.
 9. The method of claim 8, wherein said mutagenesisincludes producing fragments of the pG1 promoter which are selected fromthe group consisting of pG1a consisting of the nucleotide sequence ofSEQ ID NO: 41, pG1b consisting of the nucleotide sequence of SEQ ID NO:42, pG1c consisting of the nucleotide sequence of SEQ ID NO: 43, pG1dconsisting of the nucleotide sequence of SEQ ID NO: 44, pG1e consistingof the nucleotide sequence of SEQ ID NO: 45 and pG1f consisting of thenucleotide sequence of SEQ ID NO:
 46. 10. The method of claim 1, whereinin step a) said mutagenesis produces variants having at least 90%sequence identity to the respective promoter nucleotide sequence. 11.The method of claim 1, wherein the POI is a heterologous proteinselected from the group consisting of therapeutic proteins, antibodiesor antigen-binding fragments thereof, enzymes, peptides, proteinantibiotics, toxin fusion proteins, carbohydrate-protein conjugates,structural proteins, regulatory proteins, vaccines, vaccine likeproteins or particles, process enzymes, growth factors, hormones andcytokines.
 12. The method of claim 1, wherein the cell line is selectedfrom the group consisting of mammalian, insect, yeast, filamentous fungiand plant cell lines.